Antibodies

ABSTRACT

The use of an ScFv Ab (ScFv Ab) capable of recognizing a disease associated molecule (DAM) in the manufacture of a medicament for the prevention and/or treatment of a disease condition associated with a DAM is described. The ScFv Ab has therapeutic, diagnostic and prognostic applications.

REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE

This application is a continuation-in-part of international applicationPCT/GB00/04317, filed Nov. 13, 2000, designating the U.S., and publishedon May 25, 2001 as WO 01/36486, which claims priority fromPCT/GB99/03859, filed Nov. 18, 1999, Great Britian Application No.0003527.9, filed Feb. 15, 2000, and Great Britian Application No.0005071.6, filed Mar. 2, 2000. All of the above-mentioned applications,as well as all documents cited herein and documents referenced or citedin documents cited herein, are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to antibodies.

In particular, the present invention relates to antibodies thatrecognise a disease associated molecule (DAM).

More particularly, the present invention relates in vitro and in vivo/exvivo applications of these antibodies in the diagnosis and treatment ofdiseases associated with a DAM.

BACKGROUND TO THE INVENTION

In certain disease states, a derangement of cellular metabolism canaffect the level of expression of one or more DAMs. In somecircumstances, this cellular derangement may lead to a change in thelevels of expression of the DAM. Thus, each disease causing agent ordisease state may have associated with it a DAM which may be crucial inthe immune recognition and/or the elimination and/or control of adisease causing agent or disease state in a host organism. In this way,the DAM may be capable of acting as a marker not only for the diagnosisof disease states but also for the accurate staging of the diseaseprofile so that the appropriate therapy may be designed.

A particular example of DAMs which have been well characterised includethe tumour-associated antigens (TAAs). A number of oncofoetal ortumour-associated antigens (TAAs) have been identified and characterisedin human and animal tumours.

These TAAs include carcinoembryonic antigen (CEA), TAG72, c-erB2,(underglycosylated) MUC-1 and p53, epithelial glycoprotein-2 antigen(EGP-2; also known as EGP40, Ep-CAM, KSA, C017-1A or GA733-2) and the5T4 antigen. In general, TAAs are antigens which are expressed duringfoetal development but which are downregulated in adult cells, and arethus normally absent or present only at very low levels in adults.However, during tumourigenesis, tumour cells have been observed toresume expression of TAAs. Thus, it is thought that malignant cells maybe distinguished from their non-malignant counterparts by resumption ofexpression of TAAs. Consequently, application of TAAs for (i) in vitroand/or in vivo/ex vivo diagnosis of tumour disorders; (ii) for imagingand/or immunotherapy of cancer has been suggested and (iii) asindicators of progression of tumour associated disease;

In order to mount a humoral and/or cellular immune response against aparticular disease, the host immune system must come in contact with aDAM. In addition to recognising foreign antigens, T cells often needadditional stimulation to become fully activated. It is now becomingapparent that two signals are required for activation of naive T-cellsby antigen bearing target cells. One signal is an antigen specificsignal, delivered through the T-cell receptor and the second signal isan antigen independent or co-stimulatory signal leading to lymphokineproducts. These additional signals are delivered through other receptors(such as CD28 and CD40L) on the T cell that interact with ligands (suchas B7 and CD40) which are present on professional antigen presentingcells (APCs), such as dendritic cells and macrophages, but which areabsent from other cells. These co-stimulatory ligands are often referredto as co-stimulatory molecules.

By way of example, the B7 family (namely B7.1, B7.2, and possibly B7.3)represent a recently discovered, but important group of co-stimulatorymolecules. B7.1 and B7.2 are both member of the Ig gene superfamily. Ifa T lymphocyte encounters an antigen alone, without co-stimulation byB7, it will respond with either anergy, or apoptosis (programmed celldeath). If the co-stimulatory signal is provided it will respond withclonal expansion against the target antigen. No significantamplification of the immune response against a given antigen is thoughtto occur without co-stimulation (June et al (Immunology Today15:321-331, 1994); Chen et al(Immunology Today 14:483-486); Townsend etal (Science 259:368-370)). Freeman et al(J. Immunol. 143:2714-2722,1989). Azuma et al(Nature 366:76-79, 1993). Thus, it has been postulatedthat one method for stimulating immune recognition of diseased cellswhich are poorly immunogenic would be to enhance antigen presentationand co-stimulation of lymphocytes in the presence of the DAM.

By way of example, it has been shown that disease states such as cancer,established tumours may be poorly immunogenic despite the fact that theycommonly express DAMs. Transfection of the genes encoding B7-1 and B7-2,either alone or in combination with cytokines, have been shown toenhance the development of immunity to experimental tumours in animalmodels (e.g. Leong et al. 1997 Int. J. Cancer 71: 476-482; Zitvogel etal. 1996 Eur. J. Immunol. 26:1335-1341; Cayeux et al. 1997 J. Immunol158:2834-2841). However, in translating these results into a practicaltreatment for human cancer, there are a number of significant problemsto be overcome. A major problem in such studies has been the need todeliver B7 genes in vivo to a large number of cells of the tumour toachieve efficacy. A second problem has been the selective targetexpression of B7 to the tumour cells to avoid inappropriate immune cellactivation directed against other cell types. Some solutions to theseproblems have been addressed in WO 98/55607 where a tumour interactingprotein (TIP) such as a tumour binding protein (TBP) has been used toselectively target a co-stimulatory molecule to tumour cells.

Recombinant DNA technologies have been applied to develop antibodiesthat recognise DAMs (Hoogenboom et al 1998 Immunotechnology 4: 1-20; andWinter 1998 FEBS Lett 458: 92-94. Recently, there has been considerableinterest in using antibody gene libraries to generating antibodies, suchas a single chain antibody (ScFv Abs). It is well known that in certaincircumstances, there are advantages of using ScFv Abs, rather than wholeantibodies. The smaller size of the fragments allows for rapidclearance, and may lead to improved tumour to non-tumour ratios.However, many efforts have failed to produce ScFv Abs of highspecificity. Moreover, whole IgGs are regarded as a better format fortherapeutic Mabs than ScFc Abs as they are regarded as having anextended serum half life (see Vaughan et al 1998, Nature Biotech 16:535-539).

The present invention seeks to provide an ScFv Ab raised against a DAMwhich is useful in the treatment of disease conditions associated with aDAM.

SUMMARY ASPECTS OF THE PRESENT INVENTION

The present invention provides an ScFv Ab (ScFv Ab), capable ofrecognising a DAM and having a therapeutic effect in diseases associatedwith a DAM. This ScFv Ab can be directly administered either as apeptide (synthetically or genetically expressed) or as “naked DNA” (forexample, in a plasmid) or via a delivery vehicle such as a viral vectorcomprising the nucleotide sequence encoding the ScFv Ab. For some cases,this ScFv Ab may be more efficacious than a ScFv Ab fused to an secretedco-stimulatory molecule (SCM) such as B7 or IgG. Using an ScFv Ab wasnot an obvious choice as a therapeutic agent, for the treatment ofdiseases such as cancer, especially as one would expect that a fusionprotein comprising a SCM fused to an ScFv would perform better than anScFv alone.

The present invention is advantageous for the following reasons:

(i) it provides an ScFv Ab capable of recognising a DAM. For some cases,it has a greater therapeutic effect than an ScFv Ab which is fused to aSCM such as B7 or an immunoglobulin such as IgG;

(ii) it provides a high affinity ScFv Ab which has applications in:

(a) in vitro and in vivo/ex vivo diagnosis and therapy;

(b) imaging and the treatment of cells expressing the a DAM;

(c) prevention and/or treatment of different human diseases such ascarcinomas when the ScFv Ab is used either alone or in combination withsuitable diagnostic and/or therapeutically useful agents;

(d) studies relating to the isolation and/or purification of a DAM towhich the ScFv Abs specifically binds; and

(e) providing building blocks for further rational therapeutic ScFv Abdesign and screens for ScFv Abs capable of binding to target DAMs and/orscreens for DAMs capable of binding to target ScFv Abs.

DETAILED ASPECTS OF THE INVENTION

Other aspects of the present invention are presented in the accompanyingclaims and in the following description and drawings. These aspects arepresented under separate section headings. However, it is to beunderstood that the teachings under each section are not necessarilylimited to that particular section heading.

ScFv Antibody

In one aspect, the present invention provides a recombinant ScFv Ab thatrecognises a DAM.

As used herein, the term “ScFv Ab” means an antibody capable ofrecognising a DAM antigen which has a light chain variable region (VL)and a heavy chain variable (VH) region. The VH and VL partner domainsare typically linked/joined via a flexible oligopeptide/peptide linker.The VH and VL partner domains may be connected in the order of VHfollowed by VL or VL followed by VH. Typically, the the sequences may beconnected via a linker sequence in the order VH-linker-VL orVL-linker-VH. As used herein, the term includes fragments ofproteolytically-cleaved or recombinantly-prepared portions of an ScFv Abmolecule that are capable of selectively reacting with or recognising aDAM. Non limiting examples of such proteolytic and/or recombinantfragments include chimeric ScFv antibodies which, for the purposes ofthis invention, may refer to an ScFv Ab having either a or both heavyand light chain variable regions (VH and VL) encoded by a nucleotidesequence derivable from a mammalian immunoglobulin gene other than ahuman immunoglobulin gene and either a or both heavy and light chainencoded by a nucleotide sequence derivable from a human immunoglobulingene. The ScFv Ab may be covalently or non-covalently linked to anotherentity (such as another ScFv Ab) to form antibodies having two or morebinding sites. For example, one ScFv Ab could bind to to a DAM, such as5T4, and the second ScFv Ab could bind to an immune enhancer molecule.

In accordance with the present invention, reference to the term “ScFvAb” includes but is not limited to reference to the peptide per se alsoas well the peptide as part of a fusion protein as well as thenucleotide sequence encoding the peptide and/or the nucleotide sequenceencoding the fusion protein. The peptide per se and/or fusion proteinmay be a synthetic peptide. Alternatively, the peptide and/or fusionprotein may be a genetically expressed/recombinant peptide/fusionprotein. For some applications, the term “ScFv Ab means peptide per se.The term “ScFv Ab” also includes an ScFv Ab with a secretion leader (L)sequence which is designated herein as LScFv.

As used herein, the term “variable region” refers to the variableregion, or domain, of the light chain (VL) and heavy chain (VH) whichcontain the determinants for binding recognition specificity and for theoverall affinity of the ScFv Ab for a DAM. The variable domains of eachpair of light (VL) and heavy chains (VH) are involved in antigenrecognition and form the antigen binding site. The domains of the lightand heavy chains have the same general structure and each domain hasfour framework (FR) regions, whose sequences are relatively conserved,connected by three complementarity determining regions (CDRs). The FRregions maintain the structural integrity of the variable domain. TheCDRs are the polypeptide segments within the variable domain thatmediate binding of an antigen such as a DAM.

Preferably the affinity (K_(D)) of the ScFv Ab of the present inventionfor the 5T4 antigen is from about 5×10⁻¹⁰ to about 10×10⁻¹⁰.

Preferably the affinity (K_(D)) of the ScFv Ab of the present inventionfor the 5T4 antigen is from about 6×10⁻¹⁰ to about 9×10⁻¹⁰.

Preferably the affinity (K_(D)) of the ScFv Ab of the present inventionfor the 5T4 antigen is from about 7×10⁻¹⁰ to about 8×10⁻¹⁰.

Preferably the affinity (K_(D)) of the ScFv Ab of the present inventionfor the 5T4 antigen is about 7.9×10⁻¹⁰. The K_(D) of the ScFvAb ismeasured using BIAevaluation software (Pharmacia).

As used herein, the term “off-rate” means the dissociation rate(k_(off)) of a ScFv Ab from an antigen. In the context of the presentinvention, it is measured using BIAevaluation software (Pharmacia). Alow off rate is desirable as it reflects the affinity of an Fab fragmentfor an antigen such as a DAM.

As used herein, the term “affinity” is defined in terms of thedissociation rate or off-rate (k_(off)) of a ScFv Ab from a DAM antigen.The lower the off-rate the higher the affinity that a ScFv Ab has for anantigen such as a DAM.

DAM

As used herein, the term “DAM” can include but is not limited tobiological response modifiers which include but are not limited toimmunomodulators, cytokines, growth factors, cell surface receptors,hormones, circulatory molecule, inflammatory cytokines, and pathogenicagents such a viruses, bacteria, parasites or yeast. Examples of thesebiological response modifiers include but are not limited to ApoE,Apo-SAA, BDNF, Cardiotrophin-1, EGF, ENA-78, Eotaxin, Eotaxin-2,Exodus-2, FGF-acidic, FGF-basic, fibroblast growth factor-10 (Marshall1998 Nature Biotechnology 16: 129), FLT3 ligand (Kimura et al. (1997),Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-β1, insulin, IFN-γ, IGF-I,IGF-II, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16,IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocyte growthfactor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitorysubstance, monocyte colony inhibitory factor, monocyte attractantprotein (Marshall 1998 ibid), M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α,MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-i(MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4,Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β,SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3,tumour necrosis factor (TNF), TNF-α, TNF-β, TNIL-1, TPO, VEGF, GCP-2,GRO/MGSA, GRO-β, and GRO-γ.

Examples of pathogenic agents can include but are not limited toviruses, bacteria and parasites and yeasts. By way of example,pathogenic viruses include but are not limited to human immunodeficiencyvirus (HIV), influenza, herpes simplex, human papilloma virus, equineencephalitis virus, hepatitis, feline leukaemia virus, canine distemperand rabies virus, influenza, poxviruses, fowl pox virus (FPV), canarypoxvirus, entomopox virus, vaccinia virus deficient in a DNA replicationenzyme, Alphavirus, adenovirus, herpesvirus, Venezuelan equineencephalitis virus (VEE). Examples of pathogenic bacteria can includebut are not limited to Chlamydia, Mycobacteria, Plasmodium Falciparum,Legioniella, Pseudomonas aeruginosa, Salmonella typhimurium,Streptococcus pyogenes, Neisseria gonorrheae, Corynebacteriumdiphtheriae, Clostridium tetani, Vibrio cholerae, Listeriamonocytogenes, Clostridium perfringens, Escherichia coli, Yersiniapestis, Streptococcus pneumoniae and S. Typhimurium Examples ofpathogenic parasites include but are not limited to Trypanosoma,Trypanosoma cruzi, Leishmania, Leishmania donovani, L. tropica, L.mexicana, L. Braziliensis, Giardia, Giardia lamblia, Trichomonas,Entamoeba, Naegleria, Acanthamoeba, Acanthamoeba castellanii, A.culbertsoni and other species, Plasmodium, Toxoplasma, Toxoplasmagondii, Cryptosporidium, Cryptosporidium parvum, Isospora, Isosporabelli, Naegleria, Naegleria fowleri, Balantidium, Balantidium coli,Babesia, Schistosoma, Toxiplasma and Toxocara canis. Examples ofpathogenic yeasts include Aspergillus and invasive Candida. In apreferred embodiment the pathogenic microorganism is an intracellularorganism.

Preferably the DAM is an intracellular pathogenic agent.

Preferably the DAM is a disease associated cell surface molecule(DACSM).

In accordance with the present invention the DACSM can include but isnot limited to a receptor for adhesive proteins such as growth factorreceptors. Examples of growth factor receptors include but are notlimited to ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, ENA-78, Eotaxin,Eotaxin-2, Exodus-2, FGF-acidic, FGF-basic, fibroblast growth factor-10(Marshall 1998 Nature Biotechnology 16: 129) FLT3 ligand (Kimura et al(1997), Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-β1, insulin, IFN-γ,IGF-I, IGF-II, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocytegrowth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerianinhibitory substance, monocyte colony inhibitory factor, monocyteattractant protein (Marshall 1998 ibid), M-CSF, MDC (67 a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.),MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitorfactor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3,NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α,SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2,TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNIL-1, TPO, VEGF,GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309. A non-exhaustive list ofgrowth factor receptors can be found on pages 392-297 Molecular Biologyand Biotechnology (Ed R A Meyers 1995 VCH Publishers Inc).; aplasminogen activator; a metalloproteinase (such as colllagenase), amucin; a glycoprotein; an antigen restricted in its tissue distribution;and/or a cell surface molecule which plays a role in tumour cell growth,migration or metastasis, (such as a 5T4 antigen, a tumour specificcarbohydrate moiety or an oncofetal antigen). The term DACSM may alsoincludes antigenic determinants.

Antigenic Determinant

As used herein, the term “antigenic determinant” refers to any antigenwhich is associated with a disease or a disorder. By way of example, theantigenic determinant may also be derived from pathogenic agentsassociated with diseased cells, such as tumour cells, which multiplyunrestrictedly in an organism and may thus lead to pathological growths.Examples of such pathogenic agents are described in Davis, B. D. etal(Microbiology, 3rd ed., Harper International Edition). The antigenicdeterminant may be an antigen and/or an immunodominant epitope on anantigen. By way of example, the antigenic determinant may include tumourassociated antigens (TAA) which may serve as targets for the host immunesystem and elicit responses which result in tumour destruction.

TAA

The term “tumour associated antigen (TAA)” is used herein to refer toany TAA or antigenic peptide thereof. The antigen being one that isexpressed by the tumour itself or cells associated with the tumour suchas parenchymal cells or those of the associated vasculature. The term“tumour associated antigen (TAA)” includes antigens that distinguish thetumour cells from their normal cellular counterparts where they may bepresent in trace amounts.

Examples of TAAs include but are not limited to MART-1 (Melanoma AntigenRecognised by T cells-1) MAGE-1, MAGE-3, 5T4, gp100, Carcinoembryonicantigen (CEA), prostate-specific antigen (PSA), MUCIN (MUC-1),tyrosinase. Particularly preferred TAAs are cell surface molecules asthese are positioned for recognition by elements of the immune systemand are excellent targets for therapy such as therapy and/orimmunotherapy. The present invention is in no way limited to antigenicdeterminants encoding the above listed TAAs. Other TAAs may beidentified, isolated and cloned by methods known in the art such asthose disclosed in U.S. Pat. No. 4,514,506.

5T4 TAA

The TAA 5T4 (see WO 89/07947) has been extensively characterised. It isa 72 kDa glycoprotein expressed widely in carcinomas, but having ahighly restricted expression pattern in normal adult tissues. It appearsto be strongly correlated to metastasis in colorectal and gastriccancer. The full nucleic acid sequence of human 5T4 is known (Myers etal., 1994 J Biol Chem 169: 9319-24).

Co-Stimulatory Molecules

In order to respond to a DAM, lymphocytes require at least two distinctsignals to activate their effector functions (Bretscher and Cohn 1970Science 169: 1042-1049; Crabtree 1989 Science 243: 355-361). The primarysignal is specific for antigen. Stimulation of the primary signal inisolation normally leads to apoptosis (programmed cell death) of thelymphocyte or leads to the establishment of a state of sustainedunresponsiveness or anergy (Weiss et al. supra). In order to achieveactivation of the lymphocyte, accessory signals are required which maybe delivered by cytokines or by cell-surface co-stimulatory ligandspresent on antigen-presenting cells (APC).

There are a number of such co-stimulatory molecules now identifiedincluding adhesion molecules, LFA-3, ICAM-1, ICAM-2. Majorco-stimulatory molecules present on APC are the members of the B7 familyincluding B7-1 (CD80), B7-2 (CD86) and B7-3. These molecules are ligandsof co-stimulatory receptors on lymphocytes including CD28 (W092/00092),probably the most significant co-stimulatory receptor for restingT-cells. Different members of the B7 family of glycoproteins may deliversubtly different signals to T-cells (Nunes et al. 1996 J. Biol. Chem.271: 1591-1598).

In one embodiment of the present invention, an ScFv Ab is used whichcomprises a secreted co-stimulatory molecule (“SCM”) with bindingaffinity for a DAM, such as a tumour antigen.

ScFv Ab Source

The ScFv Ab of the present invention is obtainable from or produced byany suitable source, whether natural or not, or it may be a syntheticScFv Ab, a semi-synthetic ScFv Ab, a mimetic, a derivatised ScFv Ab, arecombinant ScFv Ab, a fermentation optimised ScFv Ab, a fusion proteinor equivalents, mutants and derivatives thereof as long as it retainsthe required DAM binding specificity of the ScFv Ab of the presentinvention. These include a ScFv Ab with DAM binding specificity whichmay have amino acid substitutions or may have sugars or other moleculesattached to amino acid functional groups.

The term “mimetic” relates to any chemical which may be a peptide,polypeptide, antibody or other organic chemical which has the samebinding specificity as the ScFv Ab of the present invention.

The term “derivative” or “derivatised” as used herein includes chemicalmodification of an ScFv Ab. Illustrative of such modifications would bereplacement of hydrogen by an alkyl, acyl, or amino group. Preferably,the ScFv Ab includes at least a portion of which has been prepared byrecombinant DNA techniques or produced by chemical synthesis techniquesor combinations thereof.

Preferably, the ScFv Ab is prepared by the use of chemical synthesistechniques.

Chemical Synthesis Methods

The ScFv Ab of the present invention or variants, homologues,derivatives, fragments or mimetics thereof may be produced usingchemical methods to synthesize the ScFv Ab amino acid sequence, in wholeor in part. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (e.g., Creighton (1983) ProteinsStructures And Molecular Principles, WH Freeman and Co, New York N.Y.).The composition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure;Creighton, supra).

Direct synthesis of the ScFv Ab or variants, homologues, derivatives,fragments or mimetics thereof can be performed using various solid-phasetechniques (Roberge J Y et al(1995) Science 269: 202-204) and automatedsynthesis may be achieved, for example, using the ABI 43 1 A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequences obtainablefrom the ScFv Ab, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with a sequence fromother subunits, or any part thereof, to produce a variant ScFv Ab.

In an alternative embodiment of the invention, the coding sequence ofthe ScFv Ab or variants, homologues, derivatives, fragments or mimeticsthereof may be synthesized, in whole or in part, using chemical methodswell known in the art (see Caruthers M H et al(1980) Nuc Acids Res SympSer 215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-232).

Preferably the ScFv Ab of the present invention comprises the amino acidsequence set out in SEQ ID No 1 (see FIG. 1).

Preferably the ScFv Ab of the present invention comprises the amino acidsequence set out in SEQ ID No 3 (see FIG. 2).

Preferably the ScFv Ab of the present invention comprises the amino acidsequence set out in SEQ ID No 4 (see FIG. 6).

Amino Acid Sequences

As used herein, the term “amino acid sequence” refers to peptide,polypeptide sequences, protein sequences or portions thereof.

Preferably, the ScFv Ab is an isolated ScFv Ab and/or purified and/ornon-native ScFv Ab.

The ScFv Ab of the present invention may be in a substantially isolatedform. It will be understood that the protein may be mixed with carriersor diluents which will not interfere with the intended purpose of theScFv Ab and still be regarded as substantially isolated. The ScFv Ab ofthe present invention may also be in a substantially purified form, inwhich case it will generally comprise the ScFv Ab in a preparation inwhich more than 90%, e.g. 95%, 98% or 99% of the ScFv Ab in thepreparation is a peptide comprising SEQ ID No 1 or SEQ ID No 3 or SEQ IDNo 4 or variants, homologues, derivatives or fragments thereof.

Variants/Homologues/Derivatives of Amino Acid Sequences

Preferred amino acid sequences of the present invention are set out inSEQ ID No 1 or SEQ ID No 3 or SEQ ID No 4 are sequences obtainable fromthe ScFv Ab of the present invention but also include homologoussequences obtained from any source, for example related viral/bacterialproteins, cellular homologues and synthetic peptides, as well asvariants or derivatives thereof.

The present invention also provides, for the first time, the full canine5T4 amino acid and nucleic acid sequences (FIG. 26 and SEQ ID Nos 14 and15). Thus the present invention also provides

-   -   i) a canine 5T4 polypeptide having the amino acid sequence shown        in SEQ ID No 14 or a variant, homologue, fragment or derivative        thereof; and    -   ii) a nucleotide sequence capable of encoding a such canine 5T4        polypeptide. Preferably the nucleotide sequence has the sequence        shown as SED ID NO 15 or a variant homologue, fragment or        derivative thereof.

Thus, the present invention covers variants, homologues or derivativesof the amino acid sequences presented herein, as well as variants,homologues or derivatives of the nucleotide sequence coding for thoseamino acid sequences.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 75, 85 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel over at least, for example, the amino acid sequence as set out inSEQ ID No 1 or SEQ ID No 3 or SEQ ID No 4 or SEQ ID No 14 of thesequence listing herein. In particular, homology should typically beconsidered with respect to those regions of the sequence known to beessential for binding specificity (such as amino acids at positions)rather than non-essential neighbouring sequences. Although homology canalso be considered in terms of similarity (i.e. amino acid residueshaving similar chemical properties/functions), in the context of thepresent invention it is preferred to express homology in terms ofsequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program. Anew tool, called BLAST 2 Sequences is also available for comparingprotein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2):247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence has a binding specificity, preferablyhaving at least the same binding specificity as the amino acid sequenceset out in SEQ ID No 1 or SEQ ID No 3 or SEQ ID No 4 or SEQ ID NO 14 ofthe sequence listing herein.

SEQ ID No 1 or SEQ ID No 3 or SEQ ID No 4 or SEQ ID No 14 of thesequence listing herein may be modified for use in the presentinvention. Typically, modifications are made that maintain the bindingspecificity of the sequence. Amino acid substitutions may be made, forexample from 1, 2 or 3 to 10 or 20 substitutions provided that themodified sequence retains the required binding specificity. Amino acidsubstitutions may include the use of non-naturally occurring analogues.

The ScFv Ab of the present invention may also have deletions, insertionsor substitutions of amino acid residues which produce a silent changeand result in a functionally equivalent ScFv Ab. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the binding specificity ofthe ScFv Ab is retained. For example, negatively charged amino acidsinclude aspartic acid and glutamic acid; positively charged amino acidsinclude lysine and arginine; and amino acids with uncharged polar headgroups having similar hydrophilicity values include leucine, isoleucine,valine, glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine. The same also applies to the canine 5T4sequence.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

Preferably, the isolated ScFv Ab and/or purified ScFv Ab and/ornon-native ScFv Ab and/or 5T4 sequence is prepared by use of recombinanttechniques.

With regard to a fragment of the canine 5T4 sequence, preferably thefragment comprises at least one, preferably some, most preferably all ofthe amino acids 1-182 and/or 297-420 shown in SEQ ID No 14.

Nucleotide Sequences

It will be understood by a skilled person that numerous differentnucleotide sequences can encode the same ScFv Ab of the presentinvention as a result of the degeneracy of the genetic code. Inaddition, it is to be understood that skilled persons may, using routinetechniques, make nucleotide substitutions that do not affect the ScFv Abencoded by the nucleotide sequence of the present invention to reflectthe codon usage of any particular host organism in which the ScFv Ab ofthe present invention is to be expressed.

The terms “variant”, “homologue” or “derivative” in relation to thenucleotide sequence set out in SEQ ID No 5 (see FIG. 1) or SEQ ID No 7(see FIG. 2) or SEQ ID No 8 (see FIG. 6) of the present inventionincludes any substitution of, variation of, modification of, replacementof, deletion of or addition of one (or more) nucleic acid from or to thesequence providing the resultant nucleotide sequence codes for a ScFv Abhaving a binding specificity, preferably having at least the samebinding specificity as the nucleotide sequence set out in SEQ ID No 5 orSEQ ID No 7 or SEQ ID No 8 of the sequence listings of the presentinvention.

The terms “variant”, “homologue” or “derivative” in relation to thenucleotide sequence set out in SEQ ID No 15 (see FIG. 26) of the presentinvention includes any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) nucleic acidfrom or to the sequence providing the resultant nucleotide sequencecodes for a canine 5T4 polypeptide, preferably a polypeptide as set outin SEQ ID No 14 of the sequence listing of the present invention.

As indicated above, with respect to sequence homology, preferably thereis at least 75%, more preferably at least 85%, more preferably at least90% homology to the sequences shown in the sequence listing herein. Morepreferably there is at least 95%, more preferably at least 98%,homology. Nucleotide homology comparisons may be conducted as describedabove. A preferred sequence comparison program is the GCG WisconsinBestfit program described above. The default scoring matrix has a matchvalue of 10 for each identical nucleotide and −9 for each mismatch. Thedefault gap creation penalty is −50 and the default gap extensionpenalty is −3 for each nucleotide.

The present invention also encompasses nucleotide sequences that arecapable of hybridising selectively to the sequences presented herein, orany variant, fragment or derivative thereof, or to the complement of anyof the above. Nucleotide sequences are preferably at least 15nucleotides in length, more preferably at least 20, 30, 40 or 50nucleotides in length.

With regard to a fragment of the canine 5T4 sequence, preferably thefragment conprises at least one, preferably some, most preferably all ofthe nucleic acids 1-546 and/or 890-1263 shown in SEQ ID No 15.

Hybridisation

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies.

Nucleotide sequences of the invention capable of selectively hybridisingto the nucleotide sequences presented herein, or to their complement,will be generally at least 75%, preferably at least 85 or 90% and morepreferably at least 95% or 98% homologous to the correspondingnucleotide sequences presented herein over a region of at least 20,preferably at least 25 or 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides. Preferred nucleotide sequences of theinvention will comprise regions homologous to the nucleotide sequenceset out in SEQ ID No 5 or SEQ ID No 7 or SEQ ID No 8 or Seq ID No 15 ofthe sequence listings of the present invention preferably at least 80 or90% and more preferably at least 95% homologous to the nucleotidesequence set out in SEQ ID No 5 or SEQ ID No 7 or SEQ ID No 8 of thesequence listings of the present invention.

The term “selectively hybridizable” means that the nucleotide sequenceused as a probe is used under conditions where a target nucleotidesequence of the invention is found to hybridize to the probe at a levelsignificantly above background. The background hybridization may occurbecause of other nucleotide sequences present, for example, in the cDNAor genomic DNA library being screened. In this event, background impliesa level of signal generated by interaction between the probe and anon-specific DNA member of the library which is less than 10 fold,preferably less than 100 fold as intense as the specific interactionobserved with the target DNA. The intensity of interaction may bemeasured, for example, by radiolabelling the probe, e.g. with ³²P.

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical nucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0). Where the nucleotide sequence of theinvention is double-stranded, both strands of the duplex, eitherindividually or in combination, are encompassed by the presentinvention. Where the nucleotide sequence is single-stranded, it is to beunderstood that the complementary sequence of that nucleotide sequenceis also included within the scope of the present invention.

Nucleotide sequences which are not 100% homologous to the sequences ofthe present invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of sources. In addition, other viral/bacterial, or cellularhomologues particularly cellular homologues found in mammalian cells(e.g. rat, mouse, bovine and primate cells), may be obtained and suchhomologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other animal species, and probingsuch libraries with probes comprising all or part of the nucleotidesequence set out in SEQ ID No 5 or SEQ ID No 7 or SEQ ID No 8 or SEQ IDNo 15 of the sequence listings of the present invention under conditionsof medium to high stringency. Similar considerations apply to obtainingspecies homologues and allelic variants of the amino acid and/ornucleotide sequences of the present invention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used. The primers used in degeneratePCR will contain one or more degenerate positions and will be used atstringency conditions lower than those used for cloning sequences withsingle sequence primers against known sequences.

Alternatively, such nucleotide sequences may be obtained by sitedirected mutagenesis of characterised sequences, such as the nucleotidesequence set out in SEQ ID No 5 or SEQ ID No 7 or SEQ ID No 8 or SEQ IDNO 15 of the sequence listings of the present invention. This may beuseful where for example silent codon changes are required to sequencesto optimise codon preferences for a particular host cell in which thenucleotide sequences are being expressed. Other sequence changes may bedesired in order to introduce restriction enzyme recognition sites, orto alter the binding specificity of the ScFv Ab encoded by thenucleotide sequences.

The nucleotide sequences of the present invention may be used to producea primer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g. labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or the nucleotidesequences may be cloned into vectors. Such primers, probes and otherfragments will be at least 15, preferably at least 20, for example atleast 25, 30 or 40 nucleotides in length, and are also encompassed bythe term nucleotide sequence of the invention as used herein.

The nucleotide sequences such as a DNA polynucleotides and probesaccording to the invention may be produced recombinantly, synthetically,or by any means available to those of skill in the art. They may also becloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer nucleotide sequences will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the targeting sequence which itis desired to clone, bringing the primers into contact with mRNA or cDNAobtained from an animal or human cell, performing a polymerase chainreaction (PCR) under conditions which bring about amplification of thedesired region, isolating the amplified fragment (e.g. by purifying thereaction mixture on an agarose gel) and recovering the amplified DNA.The primers may be designed to contain suitable restriction enzymerecognition sites so that the amplified DNA can be cloned into asuitable cloning vector

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode substantially the same or a functionally equivalent aminoacid sequence, may be used to clone and express the ScFv Ab. As will beunderstood by those of skill in the art, it may be advantageous toproduce the ScFv Ab—encoding nucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particularprokaryotic or eukaryotic host (Murray E et al(1989) Nuc Acids Res17:477-508) can be selected, for example, to increase the rate of theScFv Ab expression or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, than transcriptsproduced from naturally occurring sequence.

In one embodiment of the present invention, the ScFv Ab is a recombinantScFv Ab.

Preferably the recombinant ScFv Ab is prepared using a genetic vector.

Vector

As it is well known in the art, a vector is a tool that allows orfaciliates the transfer of an entity from one environment to another. Inaccordance with the present invention, and by way of example, somevectors used in recombinant DNA techniques allow entities, such as asegment of DNA (such as a heterologous DNA segment, such as aheterologous cDNA segment), to be transferred into a host and/or atarget cell for the purpose of replicating the vectors comprising thenucleotide sequences of the present invention and/or expressing theproteins of the invention encoded by the nucleotide sequences of thepresent invention. Examples of vectors used in recombinant DNAtechniques include but are not limited to plasmids, chromosomes,artificial chromosomes or viruses.

The term “vector” includes expression vectors and/or transformationvectors.

The term “expression vector” means a construct capable of in vivo or invitro/ex vivo expression.

The term “transformation vector” means a construct capable of beingtransferred from one species to another.

“Naked DNA”

The vectors comprising nucleotide sequences encoding ScFv Abs of thepresent invention for use in affecting viral infections may beadministered directly as “a naked nucleic acid construct”, preferablyfurther comprising flanking sequences homologous to the host cellgenome.

As used herein, the term “naked DNA” refers to a plasmid comprising anucleotide sequences encoding a ScFv Ab of the present inventiontogether with a short promoter region to control its production. It iscalled “naked” DNA because the plasmids are not carried in any deliveryvehicle. When such a DNA plasmid enters a host cell, such as aeukaryotic cell, the proteins it encodes (such as the ScFv Ab) aretranscribed and translated within the cell.

Non-Viral Delivery

Alternatively, the vectors comprising nucleotide sequences of thepresent invention may be introduced into suitable host cells using avariety of non-viral techniques known in the art, such as transfection,transformation, electroporation and biolistic transformation.

As used herein, the term “transfection” refers to a process using anon-viral vector to deliver a gene to a target mammalian cell.

Typical transfection methods include electroporation, DNA biolistics,lipid-mediated transfection, compacted DNA-mediated transfection,liposomes, immunoliposomes, lipofectin, cationic agent-mediated,cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556),multivalent cations such as spermine, cationic lipids or polylysine,1,2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterolcomplexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) andcombinations thereof.

Uptake of naked nucleic acid constructs by mammalian cells is enhancedby several known transfection techniques for example those including theuse of transfection agents. Example of these agents include cationicagents (for example calcium phosphate and DEAE-dextran) and lipofectants(for example lipofectam™ and transfectam™). Typically, nucleic acidconstructs are mixed with the transfection agent to produce acomposition.

Viral Vectors

Alternatively, the vectors comprising nucleotide sequences of thepresent invention may be introduced into suitable host cells using avariety of viral techniques which are known in the art, such as forexample infection with recombinant viral vectors such as retroviruses,herpes simplex viruses and adenoviruses.

Preferably the vector is a recombinant viral vectors. Suitablerecombinant viral vectors include but are not limited to adenovirusvectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, aretroviral vector, lentiviral vectors, baculoviral vectors, pox viralvectors or parvovirus vectors (see Kestler et al1999 Human Gene Ther10(10):1619-32). In the case of viral vectors, gene delivery is mediatedby viral infection of a target cell.

Retroviral Vectors

Examples of retroviruses include but are not limited to: murine leukemiavirus (MLV), human immunodeficiency virus (HIV), equine infectiousanaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcomavirus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemiavirus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murinesarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avianmyelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus(AEV).

Preferred vectors for use in accordance with the present invention arerecombinant viral vectors, in particular recombinant retroviral vectors(RRV) such as lentiviral vectors.

The term “recombinant retroviral vector” (RRV) refers to a vector withsufficient retroviral genetic information to allow packaging of an RNAgenome, in the presence of packaging components, into a viral particlecapable of infecting a target cell. Infection of the target cellincludes reverse transcription and integration into the target cellgenome. The RRV carries non-viral coding sequences which are to bedelivered by the vector to the target cell. An RRV is incapable ofindependent replication to produce infectious retroviral particleswithin the final target cell. Usually the RRV lacks a functional gag-poland/or env gene and/or other genes essential for replication. The vectorof the present invention may be configured as a split-intron vector. Asplit intron vector is described in PCT patent application WO 99/15683.

A detailed list of retroviruses may be found in Coffin etal(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Lentiviral Vectors

Lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al1992 EMBO. J 11: 3053-3058;Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

Adenoviruses

In one embodiment of the present invention, the features of adenovirusesmay be combined with the genetic stability of retroviruses/lentiviruseswhich can be used to transduce target cells to become transientretroviral producer cells capable of stably infect neighbouring cells.Such retroviral producer cells which are engineered to express a ScFv Abof the present invention can be implanted in organisms such as animalsor humans for use in the treatment of disease such as cancer.

Pox Viruses

Preferred vectors for use in accordance with the present invention arerecombinant pox viral vectors such as fowl pox virus (FPV), entomopoxvirus, vaccinia virus such as NYVAC, canarypox virus, MVA or othernon-replicating viral vector systems such as those described for examplein WO 95/30018.

Hybrid Viral Vectors

In a further broad aspect, the present invention provides a hybrid viralvector system for in vivo delivery of a nucleotide sequence encoding aScFc Ab of the present invention, which system comprises one or moreprimary viral vectors which encode a secondary viral vector, the primaryvector or vectors capable of infecting a first target cell and ofexpressing therein the secondary viral vector, which secondary vector iscapable of transducing a secondary target cell.

Preferably the primary vector is obtainable from or is based on anadenoviral vector and/or the secondary viral vector is obtainable fromor is based on a retroviral vector preferably a lentiviral vector.

Targeted Vector

The term “targeted vector” refers to a vector whose ability toinfect/transfect/transduce a cell or to be expressed in a host and/ortarget cell is restricted to certain cell types within the hostorganism, usually cells having a common or similar phenotype.

Replication Vectors

The nucleotide sequences encoding the ScFv Ab of the present inventionmay be incorporated into a recombinant replicable vector. The vector maybe used to replicate the nucleotide sequence in a compatible host cell.Thus in one embodiment of the present invention, the invention providesa method of making the ScFv Ab of the present invention by introducing anucleotide sequence of the present invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell.

Expression Vector

Preferably, a nucleotide sequence of present invention which is insertedinto a vector is operably linked to a control sequence that is capableof providing for the expression of the coding sequence, such as thecoding sequence of the ScFv Ab of the present invention by the hostcell, i.e. the vector is an expression vector. The ScFv Ab produced by ahost recombinant cell may be secreted or may be containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining the ScFv Ab coding sequences can be designed with signalsequences which direct secretion of the ScFv Ab coding sequences througha particular prokaryotic or eukaryotic cell membrane.

Expression in Vitro

The vectors of the present invention may be transformed or transfectedinto a suitable host cell and/or a target cell as described below toprovide for expression of an ScFv Ab of the present invention. Thisprocess may comprise culturing a host cell and/or target celltransformed with an expression vector under conditions to provide forexpression by the vector of a coding sequence encoding the ScFv Ab andoptionally recovering the expressed ScFv Ab. The vectors may be forexample, plasmid or virus vectors provided with an origin ofreplication, optionally a promoter for the expression of the saidpolynucleotide and optionally a regulator of the promoter. The vectorsmay contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. The expression of theScFv Abs of the invention may be constitutive such that they arecontinually produced, or inducible, requiring a stimulus to initiateexpression. In the case of inducible expression, ScFv Ab production canbe initiated when required by, for example, addition of an inducersubstance to the culture medium, for example dexamethasone or IPTG.

ScFv Ab Constructs

Fusion Proteins

The ScFv Ab of the invention may also be produced as fusion proteins,for example to aid in extraction and purification. Examples of fusionprotein partners include glutathione-S-transferase (GST), 6xHis, GALA(DNA binding and/or transcriptional activation domains) andβ-galactosidase. Other examples of fusion protein partners include butare not limited to a fused recombinant ScFv Ab protein comprising anantigenic co-protein such as GST, β-galactosidase or the lipoprotein Dfrom Haemophilus influenzae which are relatively large co-proteins,which solubilise and facilitate production and purification thereof.Alternatively, the fused protein may comprise a carrier protein such asbovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Incertain embodiments of the present invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen Inc) anddescribed in Gentz et al(1989 PNAS 86: 821-824). Such fusion proteinsare readily expressable in yeast culture (as described in Mitchell etal1993 Yeast 5: 715-723) and are easily purified by affinitychromatography.

Other recombinant constructions may join the ScFv Ab coding sequence tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins (Kroll D J et al(1993) DNA Cell Biol12:441-53). Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals (Porath J (1992)Protein Expr Purif 3-0.26328 1), protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, Seattle,Wash.).

It may also be convenient to include a proteolytic cleavage site betweenthe fusion protein partner and the protein sequence of interest to allowremoval of fusion protein sequences. By way of example, a fusion proteinmay also be engineered to contain a cleavage site located between thenucleotide sequence encoding the ScFv Ab and the heterologous proteinsequence, so that the ScFv Ab may be cleaved and purified away from theheterologous moiety. The inclusion of a cleavable linker sequence suchas Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and the ScFv Ab may also be useful to facilitatepurification. Preferably the fusion protein will not hinder the bindingspecificity of the ScFv Ab comprising the amino acid sequence of thepresent invention.

In one preferred embodiment, the fusion protein comprises or encodes asecreted co-co-stimulatory molecule (SCM).

SCM Fusion Proteins

The secreted co-stimulatory molecule (SCM) of the invention may be anengineered fusion protein comprising a signal peptide for secretion frommammalian cells and at least one further domain which acts as aco-stimulatory signal to a cell of the immune system. The use ofcombinations of SCMs containing different co-stimulatory domains mayalso envisaged. The ScFv Abs comprising the SCMs may be produced byexpression of SCM-encoding genes in the autologous cells of theindividual to be treated and hence any post-translational modificationsadded to the protein by the host cell are authentic and provide fullyfunctional protein and appropriate pharmacokinetics.

WO-A-92/00092 describes truncated forms of B7-1, derived by placing atranslation stop codon before the transmembrane domain, secreted frommammalian cells. In that particular case, a heterologous signal peptidefrom the Oncostatin M gene was used. WO-A92/00092 also describes fusionproteins which contain the extracellular domain of B7-1 fused to the Fcregion of an immunoglobulin. Such molecules can bind to CD28 on T-cellsand serve to stimulate T-cell proliferation. However such stimulationoccurs only to a moderate extent unless the B7 or B7-derivative isimmobilised on a solid surface.

Gerstmayer et al. (1997 J. Immol. 158: 4584-4590) describes a fusion ofB7-2 to an ScFv specific for ErbB2 followed by a myc epitope tag andpolyhistidine tag which is secreted when expressed in the yeast Pichiapastoris. This molecule retained binding for antigen and co-stimulatedproliferation of T-cells prestimulated with PMA and IL-2. However,glycosylation of such a molecule is of the yeast type, which is likelyto lead to inappropriate pharmacokinetics in humans.

In accordance with the present invention, any suitable co-stimulatorydomain(s) may be used. By way of example, co-stimulatory domains can bechosen from extracellular portions of the B7 family of cell-surfaceglycoproteins, including B7-1, B7-2 and B7-3 or other co-stimulatorycell surface glycoproteins such as but not limited to co-stimulatoryreceptor-ligand molecules including CD2/LFA-3, LFA-1/ICAM-1 and ICAM-3.Studies have demonstrated that T cell co-stimulation by monocytes isdependent on each of two receptor ligand pathways CD2/LFA-3 andLFA-1/ICAM-1 (Van Seventer et al1991 Eur J Immunol 21: 1711-1718). Inaddition, it has been shown that ICAM-3, the third LFA-1counterreceptor, is a co-stimulatory molecule for resting and activatedT lymphocytes (Hernandez-Caselles et al1993 Eur J Immunol 23:2799-2806).

Other possible co-stimulatory molecules may include a novel glycoproteinreceptor designated SLAM, has been identified which, when engaged,potentiates T-cell expansion in a CD28-independent manner and induces aTh0/Th1 cytokine production profile (Cocks et al1995 Nature 376:260-263).

CD6, a cell surface glycoprotein, has also been shown to function as aco-stimulatory and adhesion receptor on T cells. Four CD6 isoforms(CD6a, b, c, d) have been described (Kobarg et al1997 Eur J Immunol27:.2971-2980). A role for the very late antigen (VLA-4) integrin in theactivation of human memory B cells has also been suggested (Silvy etal1997 Eur J Immunol 27: 2757-2764). Endothelial cells also provideunique co-stimulatory signals that affect the phenotype of activatedCD4+ T cells (Karmann et al1996 Eur J Immunol 26: 610-617). A B3protein, present on the surface of lipopolysaccharide-activated B cells,which can provide co-stimulation to resting T cells leading to apredominant release of interleukin-4 (IL-4) and IL-5 and negligibleamounts of IL-2 and interferon gamma has been described (Vinay et al1995J Biol Chem 270: 23429-23436). The co-expression of a novelco-stimulatory T cell antigen (A6H) on T cells and tumour cells hassuggested a possible function related to common properties of thesecells (Labuda et al1995 Int Immunol 7: 1425-1432).

In one preferred embodiment of the invention, the co-stimulatory domainis a portion of B7-1 or B7-2, more preferably the complete extracellularportion of B7-1 or B7-2.

In one preferred embodiment the ScFv Ab of the present invention isformed by expression of a novel gene encoding a fusion proteincontaining the DAM binding domain or domains and the co-stimulatorydomain or domains. In the context of the present invention, theco-stimulatory domain is fused to the ScFv. The domains can be placed inthe order (N-terminus to C-terminus): antigen-binding domain followed byco-stimulatory domain; or co-stimulatory domain followed byantigen-binding domain. Preferably, the co-stimulatory domain is placedat the N-terminus followed by the antigen-binding domain. A signalpeptide is included at the N-terminus, and may be for example thenatural signal peptide of the co-stimulatory extracellular domain. Thedifferent domains may be separated by additional sequences, which mayresult from the inclusion of convenient restriction-enzyme cleavagesites in the novel gene to facilitate its construction, or serve as apeptide spacer between the domains, or serve as a flexible peptidelinker or provide another function. Preferably the domains are separatedby a flexible linker.

Two or more different genes encoding different SCMs may be used toachieve improved co-stimulation, or both co-stimulation of naive T-cellsand induction of memory responses. For example a gene encoding an SCMcontaining the B7-1 extracellular domain may be administered with a geneencoding an SCM containing the B7-2 extracellular domain.

Quantitation of SCFV Antibody Production

Although the presence/absence of marker gene expression may suggest thatthe nucleotide sequence and/or its ScFv Ab is also present, its presenceand expression may be confirmed by routine means. For example, if theScFv Ab encoding nucleotide sequence is inserted within a marker genesequence, recombinant cells containing the ScFv Ab coding regions may beidentified by the absence of the marker gene function. Alternatively, amarker gene may be placed in tandem with a ScFv Ab encoding nucleotidesequence under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the ScFv Ab as well.

Additional methods to quantitate the expression of a particular moleculeinclude radiolabeling (Melby PC et al1993 J Immunol Methods 159:235-44)or biotinylating (Duplaa C et al1993 Anal Biochem 229-36) nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated. Quantitation ofmultiple samples may be speeded up by running the assay in an ELISAformat where the ScFv Ab of interest is presented in various dilutionsand a spectrophotometric or calorimetric response gives rapidquantitation.

Host/Target Cells

Host and/or target cells comprising nucleotide sequences of the presentinvention may be used to express the ScFv Abs of the present inventionunder in vitro, in vivo and ex vivo conditions.

The term“host cell and/or target cell” includes any cell derivable froma suitable organism which a vector is capable of transfecting ortransducing. Examples of host and/or target cells can include but arenot limited to cells capable of expressing the ScFv Ab of the presentinvention under in vitro, in vivo and ex vivo conditions. Examples ofsuch cells include but are not limited to macrophages, endothelial cellsor combinations thereof. Further examples include respiratory airwayepithelial cells, hepatocytes, muscle cells, cardiac myocytes,synoviocytes, primary mammary epithelial cess and post-mitoticallyterminally differentiated non-replicating cells such as macrophagesand/or neurons.

In a preferred embodiment, the cell is a mammalian cell.

In a highly preferred embodiment, the cell is a human cell.

The term “organism” includes any suitable organism. In a preferredembodiment, the organism is a mammal. In a highly preferred embodiment,the organism is a human.

Although the ScFv Ab of the invention may be produced using prokaryoticcells as host cells, it is preferred to use eukaryotic cells, forexample yeast, insect or mammalian cells, in particular mammalian cells.Suitable host cells include bacteria such as E. Coli, yeast, mammaliancell lines and other eukaryotic cell lines, for example insect Sf9cells.

The present invention also provides a method comprising transforming ahost and/or target cell with a or the nucleotide sequence(s) of thepresent invention.

The term “transformed cell” means a host cell and/or a target cellhaving a modified genetic structure. With the present invention, a cellhas a modified genetic structure when a vector according to the presentinvention has been introduced into the cell.

Host cells and/or a target cells may be cultured under suitableconditions which allow expression of the ScFv Ab of the invention.

The present invention also provides a method comprising culturing atransformed host cell—which cell has been transformed with a or thenucleotide sequence(s) according to the present invention underconditions suitable for the expression of the ScFv Ab encoded by saidnucleotide sequence(s).

The present invention also provides a method comprising culturing atransformed host cell—which cell has been transformed with a or thenucleotide sequence(s) according to the present invention or aderivative, homologue, variant or fragment thereof—under conditionssuitable for the expression of the ScFv Ab encoded by said nucleotidesequence(s); and then recovering said ScFv Ab from the transformed hostcell culture.

The ScFv Ab of the present invention can be extracted from host cells bya variety of techniques known in the art, including enzymatic, chemicaland/or osmotic lysis and physical disruption. The ScFv Ab may bepurified and isolated in a manner known per se.

Regulation of Expression In Vitro/Vivo/Ex Vivo

The present invention also encompasses gene therapy whereby the ScFv Abencoding nucleotide sequence(s) of the present invention is regulated invitro/in vivo/ex vivo. For example, expression regulation may beaccomplished by administering compounds that bind to the ScFv Abencoding nucleotide sequence(s) of the present invention, or controlregions associated with the ScFv Ab encoding nucleotide sequence of thepresent invention, or its corresponding RNA transcript to modify therate of transcription or translation.

Control Sequences

Control sequences operably linked to sequences encoding the ScFv Ab ofthe present invention include promoters/enhancers and other expressionregulation signals. These control sequences may be selected to becompatible with the host cell and/or target cell in which the expressionvector is designed to be used. The control sequences may be modified,for example by the addition of further transcriptional regulatoryelements to make the level of transcription directed by the controlsequences more responsive to transcriptional modulators.

Operably Linked

The term “operably linked” means that the components described are in arelationship permitting them to function in their intended manner. Aregulatory sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved undercondition compatible with the control sequences.

Preferably the nucleotide sequence of the present invention is operablylinked to a transcription unit.

The term “transcription unit(s)” as described herein are regions ofnucleic acid containing coding sequences and the signals for achievingexpression of those coding sequences independently of any other codingsequences. Thus, each transcription unit generally comprises at least apromoter, an optional enhancer and a polyadenylation signal.

Promoters

The term promoter is well-known in the art and is used in the normalsense of the art, e.g. as an RNA polymerase binding site. The termencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

The promoter is typically selected from promoters which are functionalin mammalian, cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells may be used. The promoter istypically derived from promoter sequences of viral or eukaryotic genes.For example, it may be a promoter derived from the genome of a cell inwhich expression is to occur. With respect to eukaryotic promoters, theymay be promoters that function in a ubiquitous manner (such as promotersof α-actin, β-actin, tubulin) or, alternatively, a tissue-specificmanner (such as promoters of the genes for pyruvate kinase).

Hypoxic Promoters/Enhancers

The enhancer and/or promoter may be preferentially active in a hypoxicor ischaemic or low glucose environment, such that the ScFv Ab encodingnucleotide sequence(s) is preferentially expressed in the particulartissues of interest, such as in the environment of a tumour, arthriticjoint or other sites of ischaemia. Thus, any significant biologicaleffect or deleterious effect of the ScFv Ab encoding nucleotidesequence(s) on the individual being treated may be reduced oreliminated. The enhancer element or other elements conferring regulatedexpression may be present in multiple copies. Likewise, or in addition,the enhancer and/or promoter may be preferentially active in one or morespecific cell types—such as any one or more of macrophages, endothelialcells or combinations thereof. Further examples may include but are notlimited to respiratory airway epithelial cells, hepatocytes, musclecells, cardiac myocytes, synoviocytes, primary mammary epithelial cellsand post-mitotically terminally differentiated non-replicating cellssuch as macrophages and/or neurons.

Tissue-Specific Promoters

The promoters of the present invention may be tissue-specific promoters.Examples of suitable tissue restricted promoters/enhancers are thosewhich are highly active in tumour cells such as a promoter/enhancer froma MUC1 gene, a CEA gene or a 5T4 antigen gene. Examples of temporallyrestricted promoters/enhancers are those which are responsive toischaemia and/or hypoxia, such as hypoxia response elements or thepromoter/enhancer of a grp78 or a grp94 gene. The alpha fetoprotein(AFP) promoter is also a tumour-specific promoter. One preferredpromoter-enhancer combination is a human cytomegalovirus (hCMV) majorimmediate early (MIE) promoter/enhancer combination.

Preferably the promoters of the present invention are tissue specific.That is, they are capable of driving transcription of a ScFv Ab encodingnucleotide sequence(s) in one tissue while remaining largely “silent” inother tissue types.

The term “tissue specific” means a promoter which is not restricted inactivity to a single tissue type but which nevertheless showsselectivity in that they may be active in one group of tissues and lessactive or silent in another group. A desirable characteristic of thepromoters of the present invention is that they posess a relatively lowactivity in the absence of activated hypoxia-regulated enhancerelements, even in the target tissue. One means of achieving this is touse “silencer” elements which suppress the activity of a selectedpromoter in the absence of hypoxia.

The term “hypoxia” means a condition under which a particular organ ortissue receives an inadequate supply of oxygen.

The level of expression of a or the ScFv Ab encoding nucleotidesequence(s) under the control of a particular promoter may be modulatedby manipulating the promoter region. For example, different domainswithin a promoter region may possess different gene regulatoryactivities. The roles of these different regions are typically assessedusing vector constructs having different variants of the promoter withspecific regions deleted (that is, deletion analysis). This approach maybe used to identify, for example, the smallest region capable ofconferring tissue specificity or the smallest region conferring hypoxiasensitivity.

A number of tissue specific promoters, described above, may beparticularly advantageous in practising the present invention. In mostinstances, these promoters may be isolated as convenient restrictiondigestion fragments suitable for cloning in a selected vector.Alternatively, promoter fragments may be isolated using the polymerasechain reaction. Cloning of the amplified fragments may be facilitated byincorporating restriction sites at the 5′ end of the primers.

Inducible Promoters

The promoters of the present invention may also be promoters thatrespond to specific stimuli, for example promoters that bind steroidhormone receptors. Viral promoters may also be used, for example theMoloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter,the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus(CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the heterologous gene can be regulatedduring the life-time of the cell.

Inducible means that the levels of expression obtained using thepromoter can be regulated.

Enhancer

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

The term “enhancer” includes a DNA sequence which binds to other proteincomponents of the transcription initiation complex and thus facilitatesthe initiation of transcription directed by its associated promoter.

The in vitro/in vivo/ex vivo expression of the ScFv Ab of the presentinvention may be used in combination with a protein of interest (POI) ora nucleotide sequence of interest (NOI) encoding same.

Combination With POIs/NOIs

The ScFv Ab of the present invention or nucleotide sequence encodingsame may be used in combination with a POI, such as a pro-drugactivating enzyme either directly or by vector delivery to, for example,a target cell or target tissue. Instead of or as well as beingselectively expressed in target tissues, the ScFv Ab of the presentinvention or nucleotide sequence encoding same may be used incombination with another POI such as a pro-drug activation enzyme orenzymes or with a nucleotide sequences of interest (NOI) or NOIs whichencode a pro-drug activation enzyme or enzymes. These pro-drugactivation enzyme or enzymes may have no significant effect or nodeleterious effect until the individual is treated with one or morepro-drugs upon which the appropriate pro-drug enzyme or enzymes act. Inthe presence of the active POI or NOI encoding same, treatment of anindividual with the appropriate pro-drug may lead to enhanced reductionin the disease condition such as a reduction in tumour growth orsurvival.

PRO-Drug POIs

A POI, such as a pro-drug activating enzyme, may be delivered to adisease site, such as a tumour site for the treatment of a cancer. Ineach case, a suitable pro-drug is used in the treatment of the patientin combination with the appropriate pro-drug activating enzyme. Anappropriate pro-drug may be administered in conjunction with the ScFv Abor vector comprising the nucleotide sequence encoding same. Examples ofpro-drugs include: etoposide phosphate (with alkaline phosphatase,Senter et al1988 Proc Natl Acad Sci 85: 4842-4846); 5-fluorocytosine(with cytosine deaminase, Mullen et al1994 Cancer Res 54: 1503-1506);Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerret al1990 Cancer Immunol Immunother 31: 202-206);Para-N-bis(2-chloroethyl)aminobenzoyl glutamate (with carboxypeptidaseG2); Cephalosporin nitrogen mustard carbamates (with βb-lactamase);SR4233 (with P450 Reductase); Ganciclovir (with HSV thymidine kinase,Borrelli et al1988 Proc Natl Acad Sci 85: 7572-7576); mustard pro-drugswith nitroreductase (Friedlos et al1997 J Med Chem 40: 1270-1275) andCyclophosphamide (with P450 Chen et al1996 Cancer Res 56: 1331-1340).

Examples of suitable pro-drug activation enzymes for use in theinvention include a thymidine phosphorylase which activates the5-fluoro-uracil pro-drugs capcetabine and furtulon; thymidine kinasefrom Herpes Simplex Virus which activates ganciclovir; a cytochrome P450which activates a pro-drug such as cyclophosphamide to a DNA damagingagent; and cytosine deaminase which activates 5-fluorocytosine.Preferably, a pro-drug activating enzyme of human origin is used.

POIs and NOIs

Other suitable proteins of interest (POIs) or NOIs encoding same for usein the present invention include those that are of therapeutic and/ordiagnostic application such as, but are not limited to: sequencesencoding cytokines, chemokines, hormones, antibodies, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,enzymes, immune co-stimulatory molecules, immunomodulatory molecules,anti-sense RNA, a transdominant negative mutant of a target protein, atoxin, a conditional toxin, an antigen, a tumour suppressor protein andgrowth factors, membrane proteins, vasoactive proteins and peptides,anti-viral proteins and ribozymes, and derivatives therof (such as withan associated reporter group). When included, the POIs or NOIs encodingsame may be typically operatively linked to a suitable promoter, whichmay be a promoter driving expression of a ribozyme(s), or a differentpromoter or promoters, such as in one or more specific cell types.

Bystander Effect

The POI and/or NOI encoding same may be proteins which are secreted froma cell. Alternatively the POI expression products are not secreted andare active within the cell. In either event, it is preferred for the POIexpression product to demonstrate a bystander effector or a distantbystander effect; that is the production of the expression product inone cell leading to the killing of additional, related cells, eitherneighbouring or distant (e.g. metastatic), which possess a commonphenotype.

Suitable POIs or NOIs encoding same for use in the present invention inthe treatment or prophylaxis of cancer include proteins which: destroythe target cell (for example a ribosomal toxin), act as: tumoursuppressors (such as wild-type p53); activators of anti-tumour immunemechanisms (such as cytokines, co-stimulatory molecules andimmunoglobulins); inhibitors of angiogenesis; or which provide enhanceddrug sensitivity (such as pro-drug activation enzymes); indirectlystimulate destruction of target cell by natural effector cells (forexample, strong antigen to stimulate the immune system or convert aprecursor substance to a toxic substance which destroys the target cell(for example a prodrug activating enzyme). Encoded proteins could alsodestroy bystander tumour cells (for example with secreted antitumourantibody-ribosomal toxin fusion protein), indirectly stimulatedestruction of bystander tumour cells (for example cytokines tostimulate the immune system or procoagulant proteins causing localvascular occlusion) or convert a precursor substance to a toxicsubstance which destroys bystander tumour cells (eg an enzyme whichactivates a prodrug to a diffusible drug).

Also, the delivery of NOI(s) encoding antisense transcripts or ribozymeswhich interfere with expression of cellular genes for tumour persistence(for example against aberrant myc transcripts in Burkitts lymphoma oragainst bcr-abl transcripts in chronic myeloid leukemia. The use ofcombinations of such POIs and/or NOIs encoding same is also envisaged.

Examples of hypoxia regulatable therapeutic NOIs can be found inPCT/GB95/00322 (WO-A-952 1927).

ScFv Ab Coupling

The ScFv Ab of the present invention can be coupled to other moleculesusing standard methods. The amino and carboxyl termini of ScFv Ab may beisotopically and nonisotopically labeled with many techniques, forexample radiolabeling using conventional techniques (tyrosineresidues-chloramine T, iodogen, lactoperoxidase; lysineresidues-Bolton-Hunter reagent). These coupling techniques are wellknown to those skilled in the art. The coupling technique is chosen onthe basis of the functional groups available on the amino acidsincluding, but not limited to amino, sulfhydral, carboxyl, amide,phenol, and imidazole. Various reagents used to effect these couplingsinclude among others, glutaraldehyde, diazotized benzidine,carbodiimide, and p-benzoquinone.

Chemical Coupling

The ScFv Ab of the present invention may be chemically coupled toisotopes, enzymes, carrier proteins, cytotoxic agents, fluorescentmolecules, radioactive nucleotides and other compounds for a variety ofapplications including but not limited to imaging/prognosis, diagnosisand/or therapy. The efficiency of the coupling reaction is determinedusing different techniques appropriate for the specific reaction. Forexample, radiolabeling of an ScFv Ab peptide with ¹²⁵I is accomplishedusing chloramine T and Na¹²⁵I of high specific activity. The reaction isterminated with sodium metabisulfite and the mixture is desalted ondisposable columns. The labeled peptide is eluted from the column andfractions are collected. Aliquots are removed from each fraction andradioactivity measured in a gamma counter. In this manner, the unreactedNa ¹²⁵ I is separated from the labeled ScFv Ab. The peptide fractionswith the highest specific radioactivity are stored for subsequent usesuch as analysis of the ability to bind to a ScFv Ab.

Imaging

The use of labelled ScFv Abs of the present invention with short livedisotopes enables visualization quantitation of DAM binding sites in vivousing autoradiographic, or modern radiographic or other membrane bindingtechniques such as positron emission tomography in order to locatetumours with ScFv Ab binding. sites. This application provides importantdiagnostic and/or prognostic research tools.

Conjugates

In other embodiments, the ScFv Ab of the invention is coupled to ascintigraphic radiolabel, a cytotoxic compound or radioisotope, anenzyme for converting a non-toxic prodrug into a cytotoxic drug, acompound for activating the immune system in order to target theresulting conjugate to a disease site such as a colon tumour, or acell-stimulating compound. Such conjugates have a “binding portion”,which consists of the ScFv Ab of the invention, and a “functionalportion”, which consists of the radiolabel, toxin or enzyme. DifferentScFv Abs can be synthesized for use in several applications includingbut not limited to the linkage of a ScFv Ab to cytotoxic agents fortargeted killing of cells that bind the ScFv Ab.

The ScFv Ab may alternatively be used alone in order simply to block theactivity of the DAM, particularly by physically interfering with itsbinding of another compound.

The binding portion and the functional portion of the conjugate (if alsoa peptide or poypeptide) may be linked together by any of theconventional ways of cross linking polypeptides, such as those generallydescribed in O'Sullivan et al(Anal. Biochem 1979: 100, 100-108). Forexample, one portion may be enriched with thiol groups and the otherportion reacted with a bifunctional agent capable of reacting with thosethiol groups, for example the N-hydroxysuccinimide ester of iodoaceticacid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).Amide and thioether bonds, for example achieved withm-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stablein vivo than disulphide bonds.

Alternatively, if the binding portion contains carbohydrates, such aswould be the case for an antibody or some antibody fragments, thefunctional portion may be linked via the carbohydrate portion using thelinking technology in EP 0 088 695.

The functional portion of the conjugate may be an enzyme for convertinga non-toxic prodrug into a toxic drug, for example the conjugates ofBagshawe and his colleagues (Bagshawe (1987) Br. J. Cancer 56, 531;Bagshawe et al(Br. J. Cancer 1988: 58, 700); WO 88/07378) orcyanide-releasing systems (WO 91/11201).

It may not be necessary for the whole enzyme to be present in theconjugate but, of course, the catalytic portion must be present.So-called “abzymes” may be used, where a ScFv Ab is raised to a compoundinvolved in the reaction one wishes to catalyse, usually the reactiveintermediate state. The resulting antibody can then function as anenzyme for the reaction.

The conjugate may be purified by size exclusion or affinitychromatography, and tested for dual biological activities. The antigenimmunoreactivity may be measured using an enzyme-linked immunosorbentassay (ELISA) with immobilised antigen and in a live cellradio-immunoassay. An enzyme assay may be used for β-glucosidase using asubstrate which changes in absorbance when the glucose residues arehydrolysed, such as oNPG (o-nitrophenyl-β-D-glucopyranoside), liberating2-nitrophenol which is measured spectrophotometrically at 405 nm.

The stability of the conjugate may be tested in vitro initially byincubating at 37° C. in serum, followed by size exclusion FPLC analysis.Stability in vivo can be tested in the same way in mice by analysing theserum at various times after injection of the conjugate. In addition, itis possible to radiolabel the ScFv Ab with ¹²⁵I, and the enzyme with¹³¹I before conjugation, and to determine the biodistribution of theconjugate, free ScFv Ab and free enzyme in mice.

Alternatively, the conjugate may be produced as a fusion compound byrecombinant DNA techniques whereby a length of DNA comprises respectiveregions encoding the two portions of the conjugate either adjacent toone another or separated by a region encoding a linker peptide whichdoes not destroy the desired properties of the conjugate.

Conceivably, two of the functional portions of the compound may overlapwholly or partly. The DNA is then expressed in a suitable host in knownways.

Diagnostic Kits

The present invention also includes diagnostic methods and kits fordetection and measurement of DAM in biological fluids and tissues, andfor localization of a DAM in tissues. The ScFv Ab of the presentinvention that possess high binding specificity can be used to establisheasy to use kits for rapid, reliable, sensitive, and specificmeasurement and localization of a DAM in extracts of plasma, urine,tissues, and in cell culture media. The ScFv Ab of the present inventionmay also be used in a diagnostic method and kit to permit detection ofcirculating DAMs which, in certain situations, may indicate theprogression of a disease state such as the spread of micrometastases byprimary tumours in situ.

These kits may include but are not limited to the following techniques;competitive and non-competitive assays, radioimmunoassay,bioluminescence and chemiluminescence assays, fluorometric assays,sandwich assays, immunoradiometric assays, dot blots, enzyme linkedassays including ELISA, microtiter plates, antibody coated strips ordipsticks for rapid monitoring of urine or blood, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished. Intraassay and interassay variation is established at 20%,50% and 80% points on the standard curves of displacement or activity.

One example of an assay kit commonly used in research and in the clinicis a radioimmunoassay (RIA) kit. After successful radioiodination andpurification of a ScFv Ab, the antiserum possessing the highest titer isadded at several dilutions to tubes containing a relatively constantamount of radioactivity, such as 10,000 cpm, in a suitable buffersystem. Other tubes contain buffer or preimmune serum to determine thenon-specific binding. After incubation at 4° C. for 24 hours, protein Ais added and the tubes are vortexed, incubated at room temperature for90 minutes, and centrifuged at approximately 2000-2500 times g at 4° C.to precipitate the complexes of antiserum bound to the labeled ScFv Ab.The supernatant is removed by aspiration and the radioactivity in thepellets counted in a gamma counter. The antiserum dilution that bindsapproximately 10 to 40% of the labeled ScFv Ab after subtraction of thenon-specific binding is further characterized.

Immunohistochemistry

An immunohistochemistry kit may also be used for localization of DAM intissues and cells. This immunohistochemistry kit provides instructions,a ScFv Ab, and possibly blocking serum and secondary antiserum linked toa fluorescent molecule such as fluorescein isothiocyanate, or to someother reagent used to visualize the primary antiserum.Immunohistochemistry techniques are well known to those skilled in theart. This immunohistochemistry kit permits localization of a DAM intissue sections and cultured cells using both light and electronmicroscopy. It is used for both research and clinical purposes. Forexample, tumours are biopsied or collected and tissue sections cut witha microtome to examine sites of DAM production. Such information isuseful for diagnostic and possibly therapeutic purposes in the detectionand treatment of diseases such as cancer.

Foetal Cell Analysis

The ScFv antibody and/or the canine 5T4 sequence of the presentinvention are also useful in methods for isolating foetal cells frommaternal blood. Isolation of foetal cells from maternal blood has beenproposed as a non-invasive alternative to aminocentesis (see WO97/30354).

In this embodiment of the invention the DAM may be any molecule which isexpressed at different levels on maternal and foetal cells. Preferablythe DAM is expressed exclusively on foetal cells. 5T4 is known to beexpressed at very high levels on trophoblasts. Thus an antibody against5T4 may be used to isolate trophoblasts from maternal blood. Theantibody may, for example be an ScFv according to the present invention,or an antibody which is specific for (for example, raised against) a 5T4polypeptide of a different species.

Thus the present invention also provides a method for isolating a foetalcell from maternal blood using an ScFv antibody of the presentinvention, or an anti-5T4 antibody from a different species. The canine5T4 polypeptide of the present invention is useful for generating suchcross-reactive antibodies.

The foetal cell may, for example, be a trophoblast or an erythrocyte.

The maternal/foetal cells may be from a human or an animal. Hence, themethod of the present invention may be used for medical or veterinaryapplications. In a preferred embodiment, the mother and foetus arenon-human, such that the isolation method is part of a veterinaryapplication.

The isolation process may form part of a diagnostic method. For example,the foetal cells may then be subject to biochemical or genetic sampling.Such a procedure sould be used to test for foetal abnormalities such asDowns syndrome, or to determine the sex of the foetus.

Combination Therapy

The ScFv Abs of the present invention may be used in combination withother compositions and procedures for the treatment of diseases. By wayof example, the ScFv Abs may also be used in combination withconventional treatments of diseases such as cancer. By ways of furtherexample, a tumor may be treated conventionally with surgery, radiationor chemotherapy combined with a ScFv Ab or a ScFv Ab may be subsequentlyadministered to the patient to extend the dormancy of micrometastasesand to stabilize any residual primary tumor.

ScFv Ab Delivery

The ScFv Ab can be delivered with a therapeutically effective agent atthe same moment in time and at the same site. Alternatively, the ScFv Aband the therapeutically effective agent may be delivered at a differenttime and to a different site. The ScFv Ab and the therapeuticallyeffective agent may even be delivered in the same delivery vehicle forthe prevention and/or treatment of a disease condition such as cancer.

Therapeutic strategies based on the use of the ScFv Ab include therecruitment and activation of T cells by using a fusion of an DAMreactive ScFv Ab fragment with the bacterial superantigen staphylococcalenterotoxin (Dohlsten et al1994) or by using bispecific antibodies,directed to both DAM and the T-cell CD3 antigen (Kroesen et al1994).Anti-DAM antibodies may also be conjugated to different bacterial toxinsto yield potent immunotoxins (LeMaistre et al1987; Zimmermann etal1997).

ScFv Abs may be used in combination with cytotoxic agents for theprevention and/or treatment of disease states such as angiogenesisand/or cancer. Cytotoxic agents such as ricin, linked to ScFv Ab mayprovide a tool for the destruction of cells that bind the ScFv Ab. Thesecells may be found in many locations, including but not limited to,micrometastases and primary tumours.

Screens

The ScFv Ab of the present invention or a derivative or homologuethereof and/or a cell line that expresses the ScFv Ab of the presentinvention or a derivative or homologue thereof may be used to screen foragents (such as peptides, organic or inorganic molecules) capable ofaffecting the binding specificity of the ScFv Ab.

In one embodiment, the screens of the present invention may identifyagonists and/or antagonists of the ScFv Ab of the present invention.

In another embodiment, the ScFv Ab of the present invention may be usedin a variety of drug screening techniques. The ScFv Ab employed in sucha test may be free in solution, affixed to a solid support, borne on acell surface, or located intracellularly.

The abolition of ScFv Ab binding specificity or the formation of bindingcomplexes between the ScFv Ab and the agent being tested may bemeasured.

Another technique for screening provides for high throughput screening(HTS) of agents having suitable binding affinity to the ScFv Abs and isbased upon the method described in detail in WO 84/03564.

It is expected that the assay methods of the present invention will besuitable for both small and large-scale screening of test compounds aswell as in quantitative assays.

Phage Display Screens

Phage display may be employed in the identification of agents, such as aDAM that is engageable by the ScFv Ab of the present invention. Phagedisplay is a protocol of molecular screening which utilises recombinantbacteriophage. The technology involves transforming bacteriophage with anucleotide sequence encoding an appropriate ligand (such as a candidateDAM) which capable of reacting with a target ScFv Ab (or a derivative orhomologue thereof) or the nucleotide sequence (or a derivative orhomologue thereof) encoding same. The transformed bacteriophage (whichpreferably is tethered to a solid support) expresses the appropriateligand (such as the candidate agent) and displays it on their phagecoat. The entity or entities (such as cells) bearing the target ScFv Abmolecules which recognises the candidate DAM are isolated and amplified.The successful candidate DAM is then characterised.

The targeting of cells expressing a DAM with a ScFv Ab of the presentinvention facilitates the development of ScFv Abs to modulate theactivity of cells expressing the DAM

In another embodiment of the present invention, an ScFv Ab library maybe used to screen for antibodies against specific DAMs. By way ofexample, a bacteriophage may be transformed with a nucleotide sequenceencoding an appropriate ligand (such as a candidate ScFv Ab) which iscapable of reacting with a target DAM (or a derivative or homologuethereof) or the nucleotide sequence (or a derivative or homologuethereof) encoding same. The transformed bacteriophage (which preferablyis tethered to a solid support) expresses the appropriate ligand (suchas the candidate ScFv Ab) and displays it on their phage coat. Theentity or entities (such as cells) bearing the target DAM moleculeswhich recognises the candidate ScFv Ab are isolated and amplified. Thesuccessful candidate ScFv Ab is then characterised.

By way of further example, a human ScFv fragment library called “theGriffin-1 library” has been constructed by recloning synthetic heavy andlight chain variable regions (VH and VL) from the lox library vectorsinto the phagemid vector pHEN2. Modifications in the elution andscreening procedures an result in the successful screening of phagedisplay libraries for ScFv antibodies against a large variety of DAMs(see de Bruin et al1999, Nature Biotechnology 17: 397-399).

Phage display has advantages over standard affinity ligand screeningtechnologies. The phage surface displays the candidate agent in a threedimensional configuration, more closely resembling its naturallyoccuring conformation. This allows for more specific and higher affinitybinding for screening purposes.

Assays for Mimetics

The positive identification of either a DAM or a ScFv Ab using phagedisplay may faciliate the use of combinatorial libraries to identifymimetics capable of acting in the same or a similiar manner. Suchmimetics can be administered alone or in combination with othertherapeutics for the treatment of diseases associated with the DAM ofthe present invention.

Dosage

The dosage of the ScFv Ab of the present invention will depend on thedisease state or condition being treated and other clinical factors suchas weight and condition of the human or animal and the route ofadministration of the compound. Depending upon the half-life of the ScFvAb in the particular animal or human, the ScFv Ab can be administeredbetween several times per day to once a week. It is to be understoodthat the present invention has application for both human and veterinaryuse. The methods of the present invention contemplate single as well asmultiple administrations, given either simultaneously or over anextended period of time.

Formulations

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The ScFv Ab of the present invention may be effective in preventingand/or treating diseases such as cancer related diseases. The presentinvention includes the method of treating diseases such as cancerrelated disease with an effective amount of a ScFv Ab of the presentinvention. The ScFv Ab of the present invention can be provided as asynthetic peptide or an isolated and substantially purified proteins orprotein fragments or a combination thereof in pharmaceuticallyacceptable compositions using formulation methods known to those ofordinary skill in the art. These compositions can be administered bystandard routes. These include but are not limited to: oral, rectal,ophthalmic (including intravitreal or intracameral), nasal, topical(including buccal and sublingual), intrauterine, vaginal or parenteral(including subcutaneous, intraperitoneal, intramuscular, intravenous,intradermal, intracranial, intratracheal, and epidural) transdermal,intraperitoneal, intracranial, intracerebroventricular, intracerebral,intravaginal, intrauterine, or parenteral (e.g., intravenous,intraspinal, subcutaneous or intramuscular) routes.

The ScFv Ab formulations may conveniently be presented in unit dosageform and may be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

In addition, the ScFv Abs of the present invention may be incorporatedinto biodegradable polymers allowing for sustained release of thecompound, the polymers being implanted in the vicinity of where drugdelivery is desired, for example, at the site of a tumor or implanted sothat the ScFv Ab is slowly released systemically. The biodegradablepolymers and their use are described, for example, in detail in Brem etal(J. Neurosurg 1991 74:441-446). Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of ScFv Abs throughcannulae to the site of interest, such as directly into a metastaticgrowth or into the vascular supply to that tumor.

The ScFv Abs of the present invention may be linked to cytotoxic agentswhich are infused in a manner designed to maximize delivery to thedesired location. For example, ricin-linked high affinity ScFv Abs aredelivered through a cannula into vessels supplying the target site ordirectly into the target. Such agents are also delivered in a controlledmanner through osmotic pumps coupled to infusion cannulae.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition to the ingredients, particularly mentionedabove, the formulations of the present invention may include otheragents conventional in the art having regard to the type of formulationin question.

The ScFv Ab conjugates may be administered in any suitable way, usuallyparenterally, for example intravenously or intraperitoneally, instandard sterile, non-pyrogenic formulations of diluents and carriers,for example isotonic saline (when administered intravenously). Once theScFv Ab conjugate has bound to the target cells and been cleared fromthe bloodstream (if necessary), which typically takes a day or so, thepro-drug is administered, usually as a single infused dose, or thetumour is imaged. If needed, because the ScFv Ab conjugate may beimmunogenic, cyclosporin or some other immunosuppressant can beadministered to provide a longer period for treatment but usually thiswill not be necessary.

The timing between administrations of the ScFv Ab conjugate and pro-drugmay be optimised in a routine way since disease/normal tissue ratios ofconjugate (at least following intravenous delivery) are highest afterabout 4-6 days, whereas at this time the absolute amount of conjugatebound to the DAM, in terms of percent of injected dose per gram, islower than at earlier times.

Therefore, the optimum interval between administration of the ScFv Abconjugate and the pro-drug will be a compromise between peakconcentration of the enzyme at the disease site and the bestdistribution ratio between disease and normal tissues. The dosage of theScFv Ab conjugate will be chosen by the physician according to the usualcriteria. At least in the case of methods employing a targeted enzymesuch as β-glucosidase and intravenous amygdalin as the toxic pro-drug, 1to 50 daily doses of 0.1 to 10.0 grams per square metre of body surfacearea, preferably 1.0-5.0 g/m² are likely to be appropriate. For oraltherapy, three doses per day of 0.05 to 10.0 g, preferably 1.0-5.0 g,for one to fifty days may be appropriate. The dosage of the ScFv Abconjugate will similarly be chosen according to normal criteria,particularly with reference to the type, stage and location of thedisease tissue and the weight of the patient. The duration of treatmentwill depend in part upon the rapidity and extent of any immune reactionto the ScFv Ab conjugate.

The functional portion of the ScFv Ab conjugate, when the the ScFv Abconjugate is used for diagnosis, usually comprises and may consist of aradioactive atom for scintigraphic studies, for example technetium 99m(^(99m)Tc) or iodine-123 (¹²³I, or a spin label for nuclear magneticresonance (nmr) imaging (also known as magnetic resonance imaging, mri),such as iodine-123 again, iodine-313, indium-111, fluorine-19,carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

When used in a compound for selective destruction of, for example, thetumour, the functional portion of the ScFv Ab may comprise a highlyradioactive atom, such as iodine-131, rhenium-186, rhenium-188,yttrium-90 or lead-212, which emits enough energy to destroyneighbouring cells, or a cytotoxic chemical compound such asmethotrexate, adriamicin, vinca alkaliods (vincristine, vinblastine,etoposide), daunorubicin or other intercalating agents.

The radio- or other labels may be incorporated in the ScFv Ab conjugatein known ways. For example, the peptide may be biosynthesised or may besynthesised by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as ⁹⁹mTc, ¹²³I, ¹⁸⁶Rh, ¹⁸⁸Rh and ¹¹¹In can be attached via acysteine residue in the peptide. Yttrium-90 can be attached via a lysineresidue. The IODOGEN method (Fraker et al(1978) Biochem. Biophys. Res.Commun. 80: 49-57 can be used to incorporate iodine-123. “MonoclonalAntibodies in Immunoscinigraphy” (Chatal, CRC Press 1989) describesother methods in detail.

Pharmaceutical Compositions

In one aspect, the present invention provides a pharmaceuticalcomposition, which comprises an ScFv Ab according to the presentinvention and optionally a pharmaceutically acceptable carrier, diluentor excipient (including combinations thereof).

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilizers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes.

Where the pharmaceutical composition is to be delivered mucosallythrough the gastrointestinal mucosa, it should be able to remain stableduring transit though the gastrointestinal tract; for example, it shouldbe resistant to proteolytic degradation, stable at acid pH and resistantto the detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose or chalk, or in capsules or ovules either alone orin admixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

Administration

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject and it will vary with the age,weight and response of the particular patient and severity of thecondition. The dosages below are exemplary of the average case. Therecan, of course, be individual instances where higher or lower dosageranges are merited.

The compositions (or component parts thereof) of the present inventionmay be administered orally. In addition or in the alternative thecompositions (or component parts thereof) of the present invention maybe administered by direct injection. In addition or in the alternativethe compositions (or component parts thereof) of the present inventionmay be administered topically. In addition or in the alternative thecompositions (or component parts thereof) of the present invention maybe administered by inhalation. In addition or in the alternative thecompositions (or component parts thereof) of the present invention mayalso be administered by one or more of: parenteral, mucosal,intramuscular, intravenous, subcutaneous, intraocular or transdermaladministration means, and are formulated for such administration.

By way of further example, the pharmaceutical composition of the presentinvention may be administered in accordance with a regimen of 1 to 10times per day, such as once or twice per day. The specific dose leveland frequency of dosage for any particular patient may be varied andwill depend upon a variety of factors including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the age, body weight, general health, sex, diet, modeand time of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

The term “administered” also includes but is not limited to delivery bya mucosal route, for example, as a nasal spray or aerosol for inhalationor as an ingestable solution; a parenteral route where delivery is by aninjectable form, such as, for example, an intravenous, intramuscular orsubcutaneous route.

Hence, the pharmaceutical composition of the present invention may beadministered by one or more of the following routes: oraladministration, injection (such as direct injection), topical,inhalation, parenteral administration, mucosal administration,intramuscular administration, intravenous administration, subcutaneousadministration, intraocular administration or transdermaladministration.

Diseases

Pharmaceutical compositions comprising an effective amount of a ScFv Aband/or an NOI encoding same may be used in the treatment of disorderssuch as those listed in WO-A-98/09985. For ease of reference, part ofthat list is now provided: macrophage inhibitory and/or T cellinhibitory activity and thus, anti-inflammatory activity; anti-immuneactivity, i.e. inhibitory effects against a cellular and/or humoralimmune response, including a response not associated with inflammation;diseases associated with viruses and/or other intracellular pathogens;inhibit the ability of macrophages and T cells to adhere toextracellular matrix components and fibronectin, as well as up-regulatedfas receptor expression in T cells; inhibit unwanted immune reaction andinflammation including arthritis, including rheumatoid arthritis,inflammation associated with hypersensitivity, allergic reactions,asthma, systemic lupus erythematosus, collagen diseases and otherautoimmune diseases, inflammation associated with atherosclerosis,arteriosclerosis, atherosclerotic heart disease, reperfusion injury,cardiac arrest, myocardial infarction, vascular inflammatory disorders,respiratory distress syndrome or other cardiopulmonary diseases,inflammation associated with peptic ulcer, ulcerative colitis and otherdiseases of the gastrointestinal tract, hepatic fibrosis, livercirrhosis or other hepatic diseases, thyroiditis or other glandulardiseases, glomerulonephritis or other renal and urologic diseases,otitis or other oto-rhino-laryngological diseases, dermatitis or otherdermal diseases, periodontal diseases or other dental diseases, orchitisor epididimo-orchitis, infertility, orchidal trauma or otherimmune-related testicular diseases, placental dysfunction, placentalinsufficiency, habitual abortion, eclampsia, pre-eclampsia and otherimmune and/or inflammatory-related gynaecological diseases, posterioruveitis, intermediate uveitis, anterior uveitis, conjunctivitis,chorioretinitis, uveoretinitis, optic neuritis, intraocularinflammation, e.g. retinitis or cystoid macular oedema, sympatheticophthalmia, scleritis, retinitis pigmentosa, immune and inflammatorycomponents of degenerative fondus disease, inflammatory components ofocular trauma, ocular inflammation caused by infection, proliferativevitreo-retinopathies, acute ischaemic optic neuropathy, excessivescarring, e.g. following glaucoma filtration operation, immune and/orinflammation reaction against ocular implants and other immune andinflammatory-related ophthalmic diseases, inflammation associated withautoimmune diseases or conditions or disorders where, both in thecentral nervous system (CNS) or in any other organ, immune and/orinflammation suppression would be beneficial, Parkinson's disease,complication and/or side effects from treatment of Parkinson's disease,AIDS-related dementia complex HIV-related encephalopathy, Devic'sdisease, Sydenham chorea, Alzheimer's disease and other degenerativediseases, conditions or disorders of the CNS, inflammatory components ofstokes, post-polio syndrome, immune and inflammatory components ofpsychiatric disorders, myelitis, encephalitis, subacute sclerosingpan-encephalitis, encephalomyelitis, acute neuropathy, subacuteneuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora,myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington'sdisease, amyotrophic lateral sclerosis, inflammatory components of CNScompression or CNS trauma or infections of the CNS, inflammatorycomponents of muscular atrophies and dystrophies, and immune andinflammatory related diseases, conditions or disorders of the centraland peripheral nervous systems, post-traumatic inflammation, septicshock, infectious diseases, inflammatory complications or side effectsof surgery, bone marrow transplantation or other transplantationcomplications and/or side effects, inflammatory and/or immunecomplications and side effects of gene therapy, e.g. due to infectionwith a viral carrier, or inflammation associated with AIDS, to suppressor inhibit a humoral and/or cellular immune response, to treat orameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia,by reducing the amount of monocytes or lymphocytes, for the preventionand/or treatment of graft rejection in cases of transplantation ofnatural or artificial cells, tissue and organs such as cornea, bonemarrow, organs, lenses, pacemakers, natural or artificial skin tissue.Specific cancer related disorders include but not limited to: solidtumours; blood born tumours such as leukemias; tumor metastasis; benigntumours, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis;Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;wound granulation; corornay collaterals; cerebral collaterals;arteriovenous malformations; ischeniic limb angiogenesis; neovascularglaucoma; retrolental fibroplasia; diabetic neovascularization;heliobacter related diseases, fractures, vasculogenesis, hematopoiesis,ovulation, menstruation and placentation.

FIGURES

The invention will now be further described only by way of example inwhich reference is made to the following Figures:

FIG. 1 which shows a DNA sequence (SEQ ID No 5) encoding a 5T4 ScFvdesignated 5T4ScFv.1. The sequence of the mature secreted protein (SEQID No 1) is provided.

FIG. 2 which shows a DNA sequence encoding a B7-1.5T4.1 fusion protein(SEQ ID No 7). A deduced amino acid sequence for the B7-1.5T4.1 fusionprotein (SEQ ID No 3) is also provided.

FIG. 3 a which shows a diagrammatic representation of a B7-1.5T4.1construct.

FIG. 3 b which shows a diagrammatic representation of a B7-1.5T4.2construct.

FIG. 4 which shows a DNA sequence encoding a B7-2.5T4.1 fusion protein(SEQ ID No 9). A deduced amino acid sequence for the B7-2.5T4. 1 fusionprotein (SEQ ID No 10) is also provided.

FIG. 5 which shows a B7 link ScFv sequence (SEQ ID No 11).

FIG. 6 which shows a DNA sequence encoding a Ig-5T4 fusion protein (SEQID No 8). A deduced amino acid sequence for the Ig-5T4 fusion protein(SEQ ID No 4) is also provided.

FIG. 7 which shows an ScFv-IgE sequence (SEQ ID No 12).

FIG. 8 which shows a B7-EGF sequence (SEQ ID No 13).

FIG. 9 which shows the effect of the ScFv Ab on CT26-neo tumour cellgrowth in Balb/c mice over a period of 35 days.

FIG. 10 which shows the effect of the ScFv Ab on CT26-h5T4 tumour cellgrowth in Balb/c mice over a period of 35 days.

FIG. 11 which shows the effect of the ScFv Ab on B16-neo tumour cellgrowth in Balb/c mice over a period of 35 days.

FIG. 12 which shows the effect of the ScFv Ab on B16-h5T4 tumour cellgrowth in Balb/c mice over a period of 35 days.

FIG. 13 shows ScFv constructs.

FIG. 14 shows a B7-scFv binding to the 5T4 target antigen.

FIG. 15 shows B7-scFv binding to CTLA4.

FIG. 16 shows FACS analysis of A9-5T4 (A) and A9-neo (5T4 negative) (B)cells incubated with scFv protein alone or scFv-HG1 fusion proteinfollowed by goat anti-human IgG-FITC labelled antibody.

FIG. 17 shows 5T4 scFv-Hγ1 ADCC Activity.

FIG. 18 shows pONY8.1SM

FIG. 19 shows fusion protein constructs in pONY 8.1SM

-   -   a. B7-5T4scFv    -   b. L-5T4scFv

FIG. 20 shows pKLink

FIG. 21 shows an scFv and leader sequence in pBSII

FIG. 22 shows Leader-IL-5 scFv in pONY8.1SM

FIG. 23 shows Leader HIV-gp120 scFv in pONY8.1SM

FIG. 24 shows pAdApt

FIG. 25 shows Fusion protein constructs in pAdApt

-   -   a. B7-5T4scFv    -   b. L-5T4scFv

FIG. 26 shows the canine 5T4 coding sequence

IN SLIGHTLY MORE DETAIL

FIG. 14 shows supernatants from mock transfected 293T cells or thosetransfected with the tagged B7-scFv construct were incubated with A9 5T4and A9 neo cells. Detection used FITC conjugated αHis or αMycantibodies.

FIG. 15 shows A9 5T4 and A9 neo cells which were incubated with the scFvalone, a B7-scFv construct lacking the Myc-His tag or the tagged B7-scFvconstruct. The B7.1 ligand, CTLA4-Ig was added and detection used FITCconjugated αmouse IgG.

FIG. 20 shows the pKLink—the (Gly₄Ser)₃ linker in pBluescript II SK (pBSII). The flexible linker is synthesised as two complemantaryoligonucleotides, that are annealed to give restriction enzyme overhangsand then cloned as a double stranded oligonucleotide into pBSII. Theamino acid translation of (glycine₄ serine)₃ is shown in single lettercode below the DNA sequence.

FIG. 21 shows a scFv (for example an IL-5 or HIV gp120 scFv) in PBSIIand subsequent addition of the leader sequence. In this example the VHis amplified with additional Spe I and Mfe I restriction sites at the 5′end and an additional Age I site at the 3′ end. The Spe I and Age Isites are used to clone into pKlink. The VL is amplified with anadditional Bam HI restriction site at the 5′ end and an additional EcoRI site at the 3′end, which are used for cloning into pKlink. The leadersignal peptide is synthesised as two complemantary oligonucleotides,that are annealed to give restriction enzyme overhangs and then clonedas a double stranded oligonucleotide between the Spe I and Mfe I sitesat the 5′ end of the scFv cDNA. The Kozak sequence including the ATGstart codon (underlined) is in bold and italics.

FIG. 26 shows the canine 5T4 coding sequence. A mongrel genomic libraryin λ dash was screened with a probe made from h5T4 cDNA. Positive cloneswere identified and sequenced.

EXAMPLES Example 1 Construction of 5T4 ScFv Ab and Retroviral—VectorDelivery to Tumour

The cDNA encoding the murine 5T4 monoclonal antibody is cloned andsequenced by standard techniques (Antibody engineering: a practicalapproach ed McCafferty et al. 1996 OUP). The sequence of the variableregion of the antibody can be used to construct a ScFv antibody. Thecoding sequence of a 5T4 ScFv, called 5T4ScFv. 1 (SEQ ID No 1), is shownin FIG. 1. In this molecule, the DNA sequence encodes the VH from themouse 5T4 monoclonal antibody followed by a 15 amino acid flexiblelinker and the VL region of the mouse 5T4 antibody. The flexible linkerencodes 3 copies of the amino-acid sequence gly-gly-gly-gly-ser and theDNA sequence similarity between the repeats has been minimised to avoidthe risk of recombination between the repeats when plasmids containingthem are grown in E. coli.

DNA Cassettes

Cassette 1—Translation Initiation Signal and Signal Peptide

In order to achieve correct translation initiation and secretion frommammalian cells, the following sequence is used(SEQ ID NO 16):

(SEQ ID NO:38) AagcttCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCC

This contains a convenient HindIII restriction site for cloning intoexpression vectors (lower case), the consensus translation initiationsignal for mammalian cells (ANNATGPu) and the coding sequence for asignal peptide sequence from an immunoglobulin gene.

Cassette 2-scFv

The sequence of the secreted portion of the 5T4ScFv.1 is shown inFIG. 1. This molecule can be represented as Vh-(gly₄-ser)₃ linker-V1.

The 5T4 ScFv2 Ab consists of the 5T4 variable region sequences connectedin the order V1—flexible linker Vh. In this case the linker encodes the20 amino-acid peptide (gly₄-ser)₄. A longer linker improves assembly ofthe ScFv when the V-region segments are in this order. (Pluckthun etalin Antibody Engineering: a practical approach, ed McCafferty et al.1996 OUP).

Expression of a 5T4 Specific ScFv

For expression of a 5T4-specific ScFv in human cells, the codingsequence is inserted into the vector pCIneo (Promega) under the controlof a strong promoter and polyadenylation signal. The translationinitiation signal and immunoglobulin leader (signal peptide) sequencefrom Cassette 1 at the 5′end of the coding region ensure efficientsecretion of the ScFv from mammalian cells.

Example 2 Transfection of Macrophages/Monocytes with an ExpressionVector Encoding an ScFv Ab

Peripheral blood mononuclear cells are isolated from human peripheralblood at laboratory scale by standard techniques procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) and at large scale by elutriation (egCeprate from CellPro). Adherent cells (essentially monocytes) areenriched by adherence to plastic overnight and cells can be allowed todifferentiate along the macrophage differentiation pathway by culturingadherent cells for 1-3 weeks.

Monocytes and macrophages are transfected with an expression vectorcapable of expressing a ScFv Ab in human cells. For constitutive highlevel expression, the ScFv Ab is expressed in a vector which utilisesthe hCMV-MIE promoter-enhancer, pCI (Promega). For hypoxia-inducedexpression, the hCMV promoter is replaced by a promoter containing atleast one HRE. A suitable promoter is a truncated HSV TK promoter with 3copies of the mouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci.91: 6496-6500).

A variety of transfection methods can be used to introduce vectors intomonocytes and macrophages, including particle-mediated DNA delivery(biolistics), electroporation, cationic agent-mediated transfection (egusing Superfect, Qiagen). Each of these methods is carried out accordingto the manufacturer's instructions, taking into account the parametersto be varied to achieve optimal results as specified by the individualmanufacturer. Alternatively, viral vectors may be used such as defectiveAdenovirus vectors (Microbix Inc or Quantum Biotechnologies Inc).

Example 3 Construction of B7—ScFv Fusion Proteins

The extracellular domain of B7-1 is defined by amino-acid residues 1-215of the native human B7-1 protein. This sequence, together with itssignal peptide-encoding sequence, is used to construct secreted fusionproteins which also contain the ScFv derived from the 5T4 monoclonalantibody. The sequence of the 5T4 ScFv is given in FIG. 1.

A DNA coding sequence is constructed using standard molecular biologytechniques which encodes a fusion protein in which the N-terminus of the5T4 ScFv is fused after amino acid 215 of human B7-1. The sequence ofthis coding sequence, B7-1.5T4.1 (SEQ ID No 7) is shown in FIG. 2. Thefusion protein contains a flexible (gly-gly-gly-gly-ser) spacer betweenthe B7-1 and 5T4 ScFv sequences. The introduction of a convenient BamH1restriction site at the end of the linker insertion (beginning atnucleotide 733) also allows for further linkers to be screened foroptimal expression of bi-functional fusion protein. FIG. 3 indicates thefusion protein in diagrammatic form. It is similarly possible toconstruct B7-1.5T4.2 (FIG. 3 b) in which the ScFv is N-terminal and theB7 extracellular domain is C-terminal. In this case only the codingsequence of the mature B7-1 (without signal peptide) is required. Asignal peptide such as an immunoglobulin leader sequence is added to theN-terminus of the ScFv in this instance.

For fusion proteins which use the co-stimulatory extracellular domain ofB7-2 (Gerstmayer et al1997 J Immunol 158(10): 4584-90), the signalpeptide and extracellular domain of B7-2 is used in place of B7-1sequences. FIG. 4 shows the coding sequence of the SCM B7-2.5T4.1co-stimulatory domain. It encodes the first 225 amino acids of humanB7-2, preceded by its signal peptide, and a flexible linker (gly4-ser).The BamHI site at the end of this sequence can be used to insert thedomain upstream of the 5T4ScFv.1 (see FIG. 3). The sequence includes theB7-2 signal peptide which can serve to allow secretion of this fusionprotein in which the B7-2 domain is at the N-terminus of the fusionprotein.

Each engineered cDNA is inserted into the mammalian expression vectorpCI to allow expression in mammalian tissue culture cells. For thispurpose, a linker sequence is added to the 5′-end of the coding sequencewhich introduces a convenient restriction site for insertion into thepolylinker of pCI and adds the translation initiation signal CCACCimmediately adjacent to the first ATG codon. Constructs in pCI aretransfected into a suitable mammalian host cell line such as COS-1 toconfirm secretion of the SCM. The transcription cassette from pCI or anappropriate segment of the transcription cassette is subsequentlysub-cloned into the expression vector to be used as the gene deliverysystem for therapeutic use.

Example 4 Transfection of Macrophages/Monocytes with an ExpressionVector Encoding a ScFv Ab Comprising a Secreted Co-stimulatory Molecule(SCM)

Peripheral blood mononuclear cells are isolated from human peripheralblood at laboratory scale by standard techniques procedures (Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al. Chapter 9) and at large scale by elutriation (egCeprate from CellPro). Adherent cells (essentially monocytes) areenriched by adherence to plastic overnight and cells can be allowed todifferentiate along the macrophage differentiation pathway by culturingadherent cells for 1-3 weeks.

Monocytes and macrophages are transfected with an expression vectorcapable of expressing an ScFv Ab comprising an SCM in human cells. Forconstitutive high level expression, the SCM is expressed in a vectorwhich utilises the hCMV-MIE promoter-enhancer, pCI (Promega). Forhypoxia-induced expression, the hCMV promoter is replaced by a promotercontaining at least one HRE. A suitable promoter is a truncated HSV TKpromoter with 3 copies of the mouse PGK HRE (Firth et al. 1994 Proc.Natl. Acad. Sci. 91: 6496-6500).

A variety of transfection methods can be used to introduce vectors intomonocytes and macrophages, including particle-mediated DNA delivery(biolistics), electroporation, cationic agent-mediated transfection (egusing Superfect, Qiagen). Each of these methods is carried out accordingto the manufacturer's instructions, taking into account the parametersto be varied to achieve optimal results as specified by the individualmanufacturer. Alternatively, viral vectors may be used such as defectiveAdenovirus vectors (Microbix Inc or Quantum Biotechnologies Inc).

Example 5 Analysis of SCM Binding to CTLA-4 and 5T4-antigen ExpressingCells

The B7-1 or B7-2 domains of an ScFv Ab-SCM fusion protein are expectedto bind specifically to CD28 and CTLA-4 present on human T-cells.Binding to T-cells or Chinese hamster ovary cells transfected with humanCTLA-4 or CD28 is determined using FACS analysis as follows. 5×10⁵CTLA-4 expressing target cells or equivalent cells lacking CTLA-4(untransfected CHO cells) are incubated with 0.1 ml culture supernatantfrom COS-1 cells transiently transfected with SCM genes for 1 h at 4° C.The cells are washed and incubate with 1 mg monoclonal antibody specificfor the B7 domain (eg Mab 9E10) followed by FITC-labelled goatanti-mouse IgG (Pharmingen) and analysis by FACS.

Binding of ScFv to 5T4-antigen is similarly assessed using target cellsexpressing 5T4-antigen (5T4-transfected A9 cells) or control cells (A9).

Example 6 Analysis of Co-stimulatory Activity

An established mouse cell line of Balb/c origin such as HC11 cells istransfected with the cDNA encoding human 5T4-antigen (Myers et al. 1994J. Biol. Chem. 269; 9319-9324) inserted in the expression vector pCIneo.

Splenic T-cells from Balb/c mice are isolated by standard procedures(Johnstone and Thorpe 1996 In Immunochemistry in Practice. Blackwell.Chapter 4). T-cells are pre-stimulated by incubation for 1-2 days inmedium containing 10 ng/ml PMA (Sigma) and 100 U/ml human IL-2(Boehringer Mannheim). HC11-5T4 cells are incubated at 10⁴ cells /wellof a 96-well tissue culture tray for 2 h with up to 0.1 ml supernatantfrom COS cells transfected with SCM gene. Up to 10⁵ pre-stimulatedT-cells are added to each well, the cells are pulsed with 0.25 mCi/well³H-thymidine and incorporation of ³H-thymidine is measured using aliquid scintillation counter after 24 h.

Example 7 Analysis of co-stimulation in Animal Models

HC11 cells transfected with the human 5T4-antigen gene are grown astumours in Balb/c mice. SCM genes B7-1.5T4.1 or B7-2.5T4.1 or acombination of both genes are introduced into the tumour cells prior toimplantation and the growth of the tumours and the growth of controltumours which do not express SCM genes in vivo are monitored.

Example 8 Construction of a B7-1/ScFv, Specific for Human 5T4, FusionProtein

Standard molecular biology techniques are used to construct a fusionprotein consisting of the leader sequence and extracellular domain ofB7-1, fused via a flexible linker to the V_(H) and V_(L) of the murineMab 5T4 specific to human 5T4.

The flexible linker, used to join the extracellular domain of B7.1 andthe ScFv, was constructed by annealing two homologous oligonucleotideswith engineered 5′ Sma I and 3′ Spe I sites—using oligonucleotides

Upper (SEQ ID NO:6) 5′ GGG GGT GGT GGG AGC GGT GGT GGC GGC AGT GGC GGCGGC GGA A 3′ and lower (SEQ ID NO:16) 5′ CTA GTT CCG CCG CCG CCA CTG CCGCCA CCA CCG CTC CCA CCA CCC CC 3′

The linker is cloned into pBluescript (Stratagene) via Sma I and Spe Ito produce pLINK. The signal peptide (sp) and extracellular domain ofmurine B7.1 were ampified by PCR from pLK444-mB7.1 (supplied by R.Germain NIH, USA) via primers that introduce 5′ EcoRI and 3′ Sma Isites—primers forward

The linker is cloned into pBluescript (Stratagene) via Sma I and Spe Ito produce pLINK. The signal peptide (sp) and extracellular domain ofmurine B7.I were amplified by PCR from pLK444-mB7.I (supplied by R.Germain NIH, USA) via primers that introduce 5′ EcoRI and 3′

Sma I sites - primers forward (SEQ ID NO:17) 5′ C TCG AAT TCC ACC ATGGCT TGC AAT TGT CAG TTG ATG C 3′ reverse (SEQ ID NO:18) 5′ CTC CCC GGGCTT GCT ATC AGG AGG GTC TTC 3′

The B7.1 PCR product was cloned into pLINK via Eco RI and Sma I to formpBS/B7Link.

The V_(H) and V_(L) of the 5T4 specific ScFv was amplified via primers—

forward primer (SEQ ID NO:19) 5′ CTC ACT AGT GAG GTC CAG CTT CAG CAG TC3′ reverse primer (SEQ ID NO:20) 5′ CTC GCG GCC GCT TAC CGT TTG ATT TCCAGC TTG GTG CCT CCA CC 3′

that introduce 5′ Spe I and 3′ Not I sites from pHEN1-5T4 ScFv.PBS/B7Link was digested with Spe I and Not I and ligated with the ScFvto form OBM 233 consisting of the sequence shown as SEQ ID No. 11: B7Link ScFv sequence (FIG. 5).

This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccinia virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al1994 JBC). B7 is foundon the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads to apronounced increase in IL-2 which promotes CD8 (cytotoxic T cell)expansion (Linsley P S, Brady W, Grosmaire L, Aruffo A, Damle N K,Ledbetter J A J Exp Med 1991 Mar 1; 173(3):721-730 Binding of the B cellactivation antigen B7 to CD28 costimulates T cell proliferation and I1-2mRNA accumulation.) Tumour cells that have been B7 tranfected with B7have been shown retardation in animal models (Townsend S E, Allison J PScience 1993 15;259(5093):368-370).

Example 9 Transient Expression and Purification of B7-1/ScFv and LeaderScFv(LScFv)

For transient expression of B7-1/ScFv the human CMV expression plasmidpCIneo (Promega) was used. B7/ScFv was excised from OBM 233 by digestionwith EcoR I/Not I and cloned into pCIneo that was previously digestedwith EcoRI/Not I. Transient expression of recombinant protein is made bytransfection of 293T cells with the relevant plasmid using calciumphosphate (Profectin, Promega). Conditions used were similar to thoserecommended by the manufacturer. To reduce bovine serum contaminationserum free optimum media (Gibco BRL). After 36-48 hours transfectionsupernatants were harvested and spun through a Centriprep (Amicon, Glos.UK) 10 filter (all proteins larger than 10 kDa arepurified/concentrated) and a Centricon (Amicon) 10 filter. Supernatantsare concentrated approximately 30 fold.

For B7-1 to be biologically functional it must be able to displaybinding with one of it's natural ligands either CTLA-4 or CD28 found onthe surface of specific populations of T cells (e.g CD4+). Thebiological activity B7-1/ScFv fusion protein was analysed forsimultaneous interaction with its natural ligand CTLA-4 (in the form ofCTLA4-Ig supplied by Ancell, MN, USA) and A9 cells expressing human 5T4.Briefly: approximately. 5×10⁵ A9-h5T4 cells were incubated with 100 ulof either B7.1/ScFv or LScFv supernatant in a U bottom 96 well plate at4° C. for 1 hour. After washing cells were incubated with CTLA4-Ig(Ancell) for 1 hour. After washing, bound CTLA4-Ig was detected using anFITC conjugated anti-mouse Ig (Dako).

Results show obvious binding of CTLA-Ig with the B7-1 extracellulardomain, bound via the ScFv, to the surface of human 5T4 positive A9cells. The lack of binding activity with 5T4 negative A9 cells furtherillustrates that the interaction of B7 with CTLA4-Ig and ScFv with 5T4are specific.

Example 10 ScFv-IgG Fusion Example

Construction of ScFv-IgG

The sequence encoding a translation initiation sequence and the humanimmunoglobulin kappa light chain signal peptide is synthesised as twocomplementary single stranded oligonucleotides which when annealed alsocontain an internal Xho I site at the 5′ end and in addition leave a XbaI compatible 5′ overhang and a Pst I compatible 3′ overhang

(SEQ ID NO:21) ctagactcgagCCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTGGTA GCA ACA GCT ACA GGT GTC CAC TCC GAG GTC CAG ctgca and (SEQ ID NO:22)g CTG GAC CTC GGA GTG GAC ACC TGT AGC TGT TGC TAC CAA GAA GAG GAT GATACA GCT CCA TCC CAT GGTGGctcga gt

This is then cloned into pBluescript II (Stratagene) restricted with XbaI and Pst I to create pBSII/Leader.

The 5T4 ScFv is amplified by PCR from pHEN1 using oligonucleotides whichincorperate a Pst I site at the 5′ end of the product and a Hind III atthe 3′ end

GTC CAG CTG CAG CAG TCT GG (SEQ ID NO:23) and CG TTT GAT TTC AAG CTT GGTGC (SEQ ID NO:24)

This is then restricted with those enzymes and inserted intopBSII/Leader restricted with the same enzymes, creatingpBSII/Leader/ScFv.

The HlgG 1 constant region is amplified by PCR from the cloned geneusing oligonucleotides which incorperate a Hind III site at the 5′ endand a Xho I site at the 3′ end

(SEQ ID NO:25) gcgc AAG CTT gaa atc aaa cgg GCC TCC ACC AAG GGC CCA and(SEQ ID NO:26) gcgc ctcgag TCA TTT ACC CGG AGA CAG GG

This is then restricted with those enzymes and inserted intopBSII/Leader/ScFv restricted with the same enzymes, creatingpBSII/Leader/ScFv/HG1. The sequence for this construct is shown in theFIG. 4 (SEQ ID NO: 10).

This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccinia virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al1994 JBC). Bound IgGwill promote specific tumour destruction via a collection of mechanismscollectively known as antibody dependent cellular cytotoxicity (Munn etalCan res 1991 ibid, Primus et al1993 Cancer Res ibid).

Example 11 Construction of ScFv-IgE1 (Human IgE1 Heavy Constant Region)

A similar fusion construct of 5T4 ScFv—human IgE constant heavy chain ismade consisting of the sequence shown as FIG. 7 (SEQ ID No. 12).

This fusion construct is made by amplifying the human IgE1 constantheavy region by PCR cDNA derived from human B-cells RNA by RT andsubsequently using oligonucleotides which incorporate a Hind III site atthe 5′ end and a Xho I site at the 3′ end

(SEQ ID NO:27) gcgc AAG CTT gaa atc aaa cgg GCC TCC ACA CAG AGC CCA and(SEQ ID NO:28) gcgc ctcgag TCA TTT ACC GGG ATT TAC AGA

This is then restricted with those enzymes and inserted intopBSII/Leader/ScFv restricted with the same enzymes, creatingpBSII/Leader/ScFv/HE1.

As described above the ScFv-IgE construct can be incorporated into arecombinant viral vector for use in gene therapy of cancer e.g. injectpatient tissue directly or to transduce patient derivedmacrophages/moncytes/CD34+ cells ex vivo. The fusion protein will besecreted and will bind to tumour cells bearing the antigen that the ScFvis specific for. Binding of IgE to tumour cells should promote a stronghistamine response via activation of mast cells. This will lead to astrong inflammatory response and destruction tumour cells as is reportedfor IgE cytotoxic destruction of parasites e.g. helminth larvae (CapronM 1988 Eosinophils in diseases: receptors and mediators. In progress inallergy and clinical immunology (Proc. 13^(th) Int. Congress of Allergyand Clinical Immunology) Hogrefe & Huber Toronto p 6). Such inflammationand tumour destruction should initiate the recruitment of other immuneeffector cells. Past reports indicate that treatment with an MMTVantigen specific IgE Mab leads to protection from a tumour expressingMMTV antigen (Nagy E Istanvan B, Sehon A H 1991 Cancer Immunol.Immunotherapy vol 34:63-69).

Example 12 Construction of B7/EGF

B7—EGF Synthetic Gene.

A fusion construct of B7—EGF is made by inserting a PCR productamplified from the region of the gene encoding the mature EGF peptide(see accession number X04571) into pBS/B7 Link. This construct has thesequence shown in FIG. 8 (SEQ ID No. 13).

Using cDNA derived by RT of RNA isolated from a cell line such the 293human kidney line (ATCC: CRL1573), the DNA is amplified by PCR usingoligonucleotides containing a Spe I restriction enzyme site at theN-terminus and a stop codon and a Not I site at the C-terminus

(SEQ ID NO:29) GG ACT AGT AAT AGT GAC TCT GAA TGT CCC and (SEQ ID NO:30)ATT AGC GGC CGC TTA GCG CAG TTC CCA CCA CTT C

The resulting product is digested with those enzymes and ligated topBS/B7 Link which has been restricted with the same enzymes creatingpBS/B7 Link EGF. The B7 Link EGF cassette is then excised with Eco RIand Not I and inserted into a derivative of pHIT111 (Soneoka et al,1995, Nucl Acid Res 23; 628) which no longer carries the LacZ gene.

An alternative to using ScFv is to use growth factors that have a highaffinity to their corresponding receptor e.g. epidermal growth factorwhich binds to several receptors including erb-2 which is highlyassociated with tumourgenesis.

As described above the fusion construct can be incorporated into arecombinant viral vector for use in gene therapy e.g. inject patienttissue directly or to transduce patient derivedmacrophages/moncytes/CD34+ cells ex vivo. The fusion protein will besecreted and will bind to tumour cells bearing the erb-2 antigen.

Epidermal growth factor (EGF) will bind to its ligand erb-2 (an EGFreceptor) thus obviating the requirement of a ScFv. Erb-2 is highlyassociated with tumour cells (Hynes N E Semin Cancer Biol February1993;4(1):19-26, Amplification and over expression of the erbB-2 gene inhuman tumors: its involvement in tumor development, significance as aprognostic factor, and potential as a target for cancer therapy). B7 isfound on the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads tomassive increase in IL-2 which promotes CD8 (cytotoxic T cell) expansion(Linsley P S, Brady W, Grosmaire L, Aruffo A, Damle N K, Ledbetter J A JExp Med Mar. 1,1991;173(3):721-730 Binding of the B cell activationantigen B7 to CD28 costimulates T cell proliferation and interleukin 2mRNA accumulation.) Tumour cells that have been B7 transfected with B7have shown retardation in animal models (Townsend S E, Allison J PScience 1993 15;259(5093):368-370 Tumor rejection after directcostimulation of CD8+ T cells by B7-transfected melanoma cells). It ishas been reported that B7 will enhance the CTL response to tumourantigens specific to tumour cells thus leading to the destruction of allsuch cells.

Example 13 Production of Cell Lines Expressing Fusion Constructs

The ScFv-IgG gene was excised from pBSII/L/ScFv/hIgG1 by Xho Idigestion, and cloned into pLXSN via the Xho I site, to makepLXSN/ScFv-IgG, such that after chromosomal integration it is undertranscriptional control of the LTR. Virus was made in the human kidneycell line 293T by co-transfecting plasmids containing the MLV gap-polgenes (pCIEGPPD) and and the VSV G envelope (pRV67) using the tripleplasmid HIT system (Landau & Littman 1992 J Virol 66 5110, Soneoka Y etal1995 NAR 23:628-633). Virus is harvested after 48 hours and used totransduce BHK-21 cells (ATCC# CCL-10). Approximately 24 hourspost-transduction, transduced cells are selected by the addition of 1mg/ml G418 (Gibco BRL) to culture medium. The supernatant from positivecolonies was harvested and concentrated by centrifugation through aCentriprep (Amicon, Glos. UK) 10 filter (all proteins larger than 10 kDaare purified/concentrated) and a Centricon (Amicon) 10 filter.Supernatants were concentrated approximately 30 fold.

Other fusion proteins are cloned into pLXSN via the Xho I site andexpressed and concentrated using a similar protocol.

FACS Analysis of Fusion Protein Binding with Cells Expressing SpecificLigand

To determine if the ScFv-IgG fusion protein is specific for its antigen,human 5T4, FACS analysis of a human bladder carcinoma tumour line (EJ)or a stable murine cell line expressing h5T4, A9-h5T4 (Myers et al1994JBC) and a 5T4 negative line A9-neo was carried out. Approximately 5×10⁵A9 or EJ cells, in a round bottom 96 well plate (Falcon) were incubatedwith 100 ul of a 1:5 dilution of concentrated supernatant (as describedabove) for 1 hour at 4° C. After washing, bound protein is detectedusing 5 an anti human IgG/FITC conjugated antibody (Dako). Cells wereanalysed on a Becton Dickinson FACS machine. FACS results show thatthere is at least a 1 log shift in fluorescence activity in those 5T4positive cells treated with the ScFv-IgG construct compared to thenegative control construct consisting of the ScFv protein alone. A9 neoFACS shows that there is no non-specific binding of the ScFv componentof the fusion protein.

FACS analysis of ScFv-IgE is carried out similar to above except thatanti-human IgE-FITC (Dako) is used to detect binding of the fusionprotein.

The B7/EGF fusion protein is analysed for binding using FACS andHC11-erb-2 positive cells (Hynes et al1990). CTLA4-Ig (Ancell, USA) isused to analyse the bioactivity of the B7 component of the bound fusionprotein. Anti-mouse IgG-FITC is used to show CTLA-4 binding.

Analysis of Fusion Proteins

Facs Analysis of B7-scFv

Recombinant protein was generated by expression from a stablytransfected BHK-21 cell line as described below (to allow identificationand also purification) c-terminal to the scFV (FIG. 13B) in the plasmidpCIneo (Promega). To demonstrate that the scFv is able to bind to the5T4 antigen, supernatants from these and from mock transfected 293Tcells were added to mouse A9 cells expressing h5T4 (stable transfectantswith a h5T4/neomycin resistance expression construct). Detection usingFITC conjugated αHis or αMyc antibodies confirmed binding of the scFv tothe A9-5T4 cells but not 5T4 negative A9 neo cells indicating that thefusion construct is able to bind the target antigen (FIG. 14).

Further FACS analysis was undertaken to show that the B7-scFv protein isable to bind simultaneously the B7.1 ligand, CTLA4 and cells expressingh 5T4. A9 5T4 and A9 neo cells were incubated with the scFv alone, aB7-scFv construct lacking the Myc-His tag or the tagged B7-scFvconstruct. The B7.1 ligand, CTLA4-Ig was added and detected using FITCconjugated amouse IgG (FIG. 15). The presence or absence of the Myc-Histag made little difference to the simultaneous binding of the protein to5T4 antigen and CTLA-4.

Analysis of 5T4 scFv HIgG 1 Protein

Recombinant protein was generated by stable transfection of BHK-21 cellswith constructs containing either 5T4 scFv alone or 5T4scFv-Hgl (FIG.13A & C respectively) fusion under the control of the CMVimmediate/early promoter. FIG. 16 shows FACS analysis of mouse A9 5T4cells. The cells were incubated with cell supernatent from BHK-21 cellsexpressing either scFv alone or scFv-HG1, followed by goat anti-humanIgG—FITC labelled antibody. As can be seen the scFv-HG1 is able to bindthe 5T4 expressing cells and can be detected with the goat anti-humanIgG—FITC labelled antibody. FIG. 16b shows that this is due to thepresence of 5T4 at the cell surface since no binding is observed with A9cells that express the neomycin resistance marker, but no h5T4.

The same supernatents were used in an antibody dependent cell-mediatedcytotoxicity (ADCC) assay which demonstrated that the scFv-Hγ1 fusionprotein is able to direct lysis of A9 5T4 cells. The A9 5T4 and neo celllines were used in a chromium release assay. After labelling with ⁵¹Cr,cells were incubated with either no protein the scFv alone or thescFv-Hγ1 fusion construct. Freshly isolated peripheral blood lymphocyteswere added and incubated for 4 hours. An aliquot of supernatant wastaken for scintillation counting.

$\%{\mspace{11mu}\;}{lysis}\mspace{14mu}{was}\mspace{14mu}{calculated}\mspace{14mu}{as}\text{:}\frac{{{Test}\mspace{14mu}{Release}} - {{Spontaneous}\mspace{14mu}{Release}}}{{{{Maximum}\mspace{14mu}{Release}} - {{Spontaneous}\mspace{14mu}{Release}}}\;} \times 100$

Up to ˜40% lysis was obtained with increasing effector:target ratio whencompared to the scFv alone. The 5T4 negative cell line showed noincreased lysis (FIG. 17).

Example 14 Analysis of Efficacy in Animal Models

Human tumour—derived cell lines and tissues are cultured in vivo ingenetically immunodeficient, “nude” mice according to well establishedtechniques (see for example Strobel et al. 1997 Cancer Res. 57:1228-1232; McLeod et al. 1997 Pancreas 14: 237-248). Syngeneic mousemodels, in which a syngeneic tumour line is introduced into animmunocompetent mouse strain may also be used. These serve as suitableanimal models for evaluating gene delivery systems of the invention.Vectors or engineered cells are administered systemically or directlyinto the tumour and tumour growth is monitored in treated and untreatedanimals. This system is used to define the effective dose range of thetreatments of the invention and the most appropriate route ofadministration.

ScFv Fusion Protein In vivo Anti Tumour Efficacy Data

The purpose of the study was to test the efficacy of a series of singlechain antibody fusion proteins.

Murine mouse models, based on CT26, a chemically induced adenocarcinomaof BALB/c origin (Brittain et al., (1980) Cancer Res. 40:179-184), andon B16, a melanoma line derived from C57 B6 mice. Both the CT26 line andB16 are stably transformed to express human and murine 5T4. Mice areinjected I.V. (to induce lung nodules, CT26) or subcutaneously (CT26 andB16) to make single mass subcutaneous tumours.

Experimental Design

CT26 cells expressing human 5T4 (CT26-h5T4) and CT26-neo

Cells were pre-incubated with:

PBS, LScFv-1, LScFv-2, B7-ScFv, ScFv-Ig

LScFv-1 and 2 were expressed in a BHK cell line. LScFv-1 was purifiedvia its Histidine tag on a Nickel column and ScFv-2 was purified using afiltration system. B7-ScFv was purified from a BHK line via a His tagand ScFv-Ig was purified via a filtration column. The concentration ofeach ScFv used in the experiment was defined as the amount of proteinrequired to saturate binding of CT26-h5T4 cells in a FACS assay.

CT26-h5T4 and CT26-neo cells were pre-incubated with saturating amountsof each ScFv and incubated for 1 hour. After washing cells 5×10⁵ cellswere injected subcutaneously into the flanks of syngeneic BALB/c mice.

Tumour measurements were taken every two days and the volume calculated.

Results 14

FIG. 9: CT26-neo

There is not a significant difference between the groups studied apartfrom the treatment with LScFv-l,for which there is an approximate 3-foldreduction in tumour size compared to the PBS control 36 days aftertumour inoculation.

FIG. 10: CT26-h5T4

Tumours treated with all of the 5T4 ScFv constructs had a significanteffect on tumour growth. Four of the five mice treated with 5T4 ScFv-1were tumour free on day 36. On day 36 ScFv-1 treated tumour cellswere >60 fold smaller than tumours treated with PBS.

When similiar experiments were carried out using a mouse melanoma line(B16) engineered to express h5T4 a minimal anti-tumour effect was foundwith the ScFv constructs used (see FIGS. 11 and 12). The CT26 appear tobe more sensitive to anti-tumour immune responses induced by ScFvbinding than the B16 cells. In addition, B16 cells do not express murine5T4 whereas CT26 cells have mRNA to murine 5T4.

In summary there appears to be no benefit of fusing B7 or IgG to the 5T4specific ScFv in the CT26 and B16 murine models. In fact, we have foundin our examples that the ScFv alone is more efficacious than the ScFvfusion constructs due to its higher binding affinity (as shown inBIACORE compared to B7-ScFv). Thefore these data indicate that the ScFvalone has a significant effect on tumour retardation and immuneenhancing molecules fused to the ScFv may not be required to show aneffect on tumour retardation in the 5T4 model.

Example 15 Production of Lentiviral Vectors Expressing the FusionConstructs

B7-5T4 scFv and L-5T4 scFv cloning into pONY 8.1SM

pONY8.1SM (FIG. 18) is an EIAV vector with four unique cloning sitesdownstream of a CMV promoter. It is derivable from the vector shown inWO 99/32646, FIG. 1. pONY 8.1SM is the most minimal EIAV vector to datein terms of EIAV sequence that it contains (˜1.1 kb) and EIAV proteinsit expresses (none).

In order to clone B7-5T4 scFv and Leader-5T4 scFv (L-5T4 scFv) into pONY8.1SM, the sequences are amplified by PCR from the constructs previouslycloned into pBluescript II (see Examples 8 and 10), to incorporate anSbf I site at the 5′ end of the gene and an EcoRI site after thetermination codon, using the primers shown below. The products are thendirectly ligated to pONY 8.1SM, previously digested with the sameenzymes.

For B7-5T4 scFv the primers are as follows:

Primer 1. B7-Sbf

(SEQ ID NO:31) ATCGCCTGCAGG

ATG

CTTGCAATTGTCAG

Sbf I site=underlined

Kozak sequence=bold and italics with the ATG start codon underlined.

2. 5T4sc-RI

(SEQ ID NO:32) GCGCGAATTC

CCGTTTGATTTCCAGCTTGGT

Eco RI site=underlined

TAA stop codon=bold and italics

The resultant product is then cloned into pONY 8.1 SM to produce thefusion protein construct shown in FIG. 19 a.

For L-5T4 scFv the primers are as follows:

Primer 1. L-Sbf

(SEQ ID NO:33) ATCGCCTGCAGG

ATG

GATGGAGCTGTAT

Sbf I site=underlined

Kozak sequence=bold and italics, with the ATG start codon underlined.

Primer 2. 5T4sc-RI

(SEQ ID NO:34) GCGCGAATTC

CCGTTTGATTTCCAGCTTGGT

Eco RI site=underlined

TAA stop codon=bold and italics

The resultant product is then cloned into pONY 8.1 SM to produce theconstruct shown in FIG. 9 b.

Assembly and Cloning of scFv Specific for IL-5

The anti-IL-5 scFv is assembled by RT-PCR using material prepared from ahybridoma line such as the one expressing the humanised Mab to IL-5, SB240563 (Leckie, M J, Am. J. Respir. Crit. Care Med. 159, A624 1999).Techniques are similar to that described by Clackson et al (Geneticallyengineered monoclonal antibodies. Br J Rheumatol. 1991;30 Suppl 2:36-9).Briefly, Total RNA is prepared from SB 240563 cells. First strandsynthesis is performed using MMLV reverse transcriptase using oligo dTprimer. Template cDNAs are amplified by PCR with V_(H) and V_(L) genespecific primer pairs that include restriction enzyme sites, such asthose shown below, to allow cloning into pKLink, a pBluescript II SK(PBSII) plasmid that contains a flexible linker sequence, (Gly₄Ser)₃(FIG. 20) This forms the single chain antibody cDNA (FIG. 19). A doublestranded oligonucleotide encoding a translation initiation, Kozaksequence and the human Ig kappa light chain signal peptide forsecretion, similar to that described in the construction of the scFv to5T4 (see Example 10), is then cloned upstream of the scFv (FIG. 21).

The whole construct is then excised with Sbf I and Eco RI and clonedinto pONY 8.1 SM (FIG. 22).

Assembly and Cloning of scFv Specific for the Envelope Protein gp120 ofHIV

The anti-HIV scFv is assembled by RT-PCR using material prepared from ahybridoma line expressing a mAb to the envelope protein gp120 of HIV,such as mAb 110.3 (Conelly et al, Virology 295: 554-557, 1994.).Alternatively guided selection is used to make a humanised antibody (seeBeiboer S H et al, J Mol Biol,2000; 296:833-849) from which the scFv isthen derived. Techniques are similar to that described by Clackson et al(Genetically engineered monoclonal antibodies. Br J Rheumatol. 1991;30Suppl 2:36-9). Briefly, Total RNA is prepared from the hybridoma cells.First strand synthesis is performed using MMLV reverse transcriptaseusing oligo dT primer. Template cDNAs are amplified by PCR with V_(H)and V_(L) gene specific primer pairs that include restriction enzymesites, such as those shown below, to allow cloning into pKLink, apBluescript II SK (pBSII) plasmid that contains a flexible linkersequence, (Gly₄Ser)₃ (FIG. 20) This forms the single chain antibody cDNA(FIG. 21). A double stranded oligonucleotide encoding a translationinitiation, Kozak sequence and the human Ig kappa light chain signalpeptide for secretion, similar to that described in the construction ofthe scFv to 5T4 (see Example 10), is then cloned upstream of the scFv(FIG. 19).

The whole construct is then excised with Sbf I and Eco RI and clonedinto pONY 8.1SM (FIG. 18) to produce the construct shown in FIG. 23.

Example 16 Production of Adenoviral Vectors Expressing the FusionConstructs

Production of Recombinat Adenovirus Expressing 5T4scFv FusionConstructs, IL-5 scFv and ⁻HIV gp120 scFv.

B7-5T4 scFv and L-5T4 scFv cloning into pAdApt

An adenovirus transfer vector (pAdApt; see FIG. 24) with eight uniquecloning sites downstream of a CMV promoter is available from Crucell,Leiden, Netherlands.

In order to clone B7-5T4 scFv and Leader-5T4 scFv (L-5T4 scFv) intopAdApt the sequences are excised from the constructs previously clonedinto pBluescript II (see examples 8 and 10) and ligated into the vectoras follows:

For B7-5T4 scFv:—

The B7-scFv is digested with Xba I, filled in to give a blunt end thendigested with Eco RI. This fragment is then ligated to the pAdApt vectorpreviously digested with Hpa I and Eco RI (FIG. 25A).

For L-5T4 scFv:—

The L-5T4 scFv is excised with Xho I, filled in to give blunt ends andthen ligated to the pAdApt vector previously digested with Hpa I.Subsequent clones are then checked for the correct orientation of theL-5T4 scFv insert (FIG. 25B).

Cloning of scFv Specific for IL-5 into pAdApt

The L-scFv cloned into pBSII (see Example 13) is digested with Xba I,filled in to give a blunt end and then digested with Eco RI. The pAdAptvector is digested with Hind III filled in to give a blunt end and thendigested Eco RI. The two molecules are then ligated to give arecombinant transfer vector resembling FIG. 25B above (with theexception that the Eco RI restriction site is at the 3′ end of thefusion construct, the 5′ end of the gene abutting the filled in Hind IIIsite).

Cloning of scFv Specific for the Envelope Protein gp120 of HIV

The L-scFv cloned into pBSII (see Example 13) is digested with Xba I,filled in to give a blunt end and then digested with Eco RI. The pAdAptvector is digested with Hind III filled in to give a blunt end and thendigested Eco RI. The two molecules are then ligated to give arecombinant transfer vector resembling FIG. 25B above (with theexception that the Eco RI restriction site is at the 3′ end of thefusion construct, the 5′ end of the gene abutting the filled in Hind IIIsite).

Production of Recombinant Adenovirus Expressing the scFv FusionConstructs

To produce recombinant adenovirus expressing the scFv fusion constructs,PerC6 cells are transfected with equimolar amounts of the recombinanttransfer vector containing the fusion construct and an adenovirus Genomevector (AdEasy from Quantum Apligene, Harefield UK). Recombinant virusis then harvested as described in the Crucell protocol.

SUMMARY

The present invention therefore provides antibodies capable ofrecognising a disease associated cell surface marker (DAM). Theseantibodies may be used in the diagnosis and treatment of diseasesassociated with a DAM.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be covered by thepresent invention.

SEQUENCE LISTING PART OF THE DESCRIPTION

SEQ ID NO. 1 and SEQ ID No 5

See FIG. 1 (5T4ScFv. 1)

SEQ ID NO. 3 and SEQ ID No 7

See FIG. 2 (B7-1. 5T4ScFv. 1)

SEQ ID NO. 4 and SEQ ID No 8

See FIG. 6 (5T4ScFv. 1-IgG)

SEQ ID NO. 9 and SEQ ID No 10

See FIG. 4 (B7-2.5T4 1)

SEQ ID No 11

FIG. 5 (B7 link ScFv)

SEQ ID NO.12

FIG. 7 (ScFv-IgE)

SEQ ID NO. 13

FIG. 8 (B7-EGF)

SEQ ID NO. 14 and 15

FIG. 26 (canine 5T4 sequence)

1. An isolated nucleic acid molecule comprising the nucleotide sequencerepresented by SEQ ID No: 5 or a fragment thereof, wherein thenucleotide sequence encodes an ScFv antibody (ScFv Ab) that binds to 5T4antigen.
 2. An isolated nucleic acid molecule that comprises anucleotide sequence that is capable of hybridising under stringentconditions that include washing at 65° C. in 0.1×SSC to a fullcomplement of a polynucleotide comprising SEQ ID NO: 5; wherein thenucleotide sequence encodes a ScFv antibody that binds to 5T4 antigen.3. The isolated nucleic acid molecule nucleotide according to claim 2wherein the nucleotide sequence is operably linked to a promoter.
 4. Anisolated construct, vector, plasmid, or host cell comprising the nucleicacid molecule according to claim
 2. 5. An isolated construct, vector,plasmid, or host cell comprising the nucleic acid molecule according toclaim
 1. 6. The nucleic acid molecule according to claim 1 wherein thenucleotide sequence is operably linked to a promoter.
 7. An isolatedconstruct, vector, plasmid, or host cell comprising the nucleic acidmolecule according to claim
 6. 8. A process for preparing an ScFvantibody (ScFv Ab) capable of recognizing 5T4 antigen, said processcomprising expressing the nucleic acid molecule of claim 1 in a cell. 9.The process of claim 8, wherein said nucleic acid molecule is in aconstruct, vector or plasmid.
 10. An isolated nucleic acid moleculeencoding an ScFv antibody (ScFv Ab) comprising a nucleotide sequencerepresented by SEQ ID No: 1 or a fragment thereof, wherein the ScFv Abbinds to 5T4 antigen.
 11. An isolated construct, vector, plasmid, orhost cell comprising the nucleic acid molecule according to claim 10.12. The nucleic acid molecule according to claim 10 wherein thenucleotide sequence is operably linked to a promoter.
 13. An isolatedconstruct, vector, plasmid, or host cell comprising the nucleic acidmolecule according to claim
 12. 14. A process for preparing an ScFvantibody (ScFv Ab) capable of recognizing 5T4 antigen, said processcomprising expressing the nucleic acid molecule of claim 10 in a cell.15. The process of claim 14, wherein said nucleic acid molecule is in aconstruct, vector or plasmid.
 16. An isolated nucleic acid molecule thatcomprises a nucleotide sequence having at least 95% identity to thenucleotide sequence of SEQ ID NO: 5 or a fragment thereof, wherein thenucleic acid molecule encodes an ScFv antibody (ScFv Ab) that binds to5T4 antigen.
 17. An isolated nucleic acid molecule that comprises anucleotide sequence encoding an ScFv antibody (ScFv Ab) comprising asequence having at least 95% identity to the sequence of SEQ ID NO: 1 ora fragment thereof, wherein the ScFv Ab binds to 5T4 antigen.
 18. Theprocess according to claim 8, further comprising purifying the ScFvantibody.
 19. The process according to claim 14, further comprisingpurifying the ScFv antibody.
 20. A process for preparing an ScFvantibody (ScFv Ab) capable of recognizing 5T4 antigen, said processcomprising expressing the nucleic acid molecule of claim 2 in a cell.21. The process according to claim 20, further comprising purifying theScFv antibody.
 22. A process for preparing an ScFv antibody (ScFv Ab)capable of recognizing 5T4 antigen, said process comprising expressingthe nucleic acid molecule of claim 16 in a cell.
 23. The processaccording to claim 22, further comprising purifying the ScFv antibody.24. A process for preparing an ScFv antibody (ScFv Ab) capable ofrecognizing 5T4 antigen, said process comprising expressing the nucleicacid molecule of claim 17 in a cell.
 25. The process according to claim24, further comprising purifying the ScFv antibody.